mirvana probe and marker kit

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mir Vanatrade Probe amp Marker Kit(Part Number AM1554)

Protocol

I Introduction A Product Description and Background Information

B Reagents Provided with the Kit and Storage

C Materials Not Provided with the Kit

D Related Products Available from Applied Biosystems

II mir Vana Probe amp Marker Kit Procedure

A Nucleic Acid Substrate Preparation and Planning

B 5 End Labeling Reaction

C Preparation of Radiolabeled Decade Markers

D Column Purification to Remove Free Nucleotides

III Troubleshooting

A Using the Control Substrates

B Troubleshooting Poor Labeling

IV Additional Procedures

A Gel Analysis of Oligonucleotides and Labeling Products

B Determining Percent Incorporation and Specific Activity

C Alkaline Hydrolysis of RNA Probes

D Gel Purification of Probe

E Precipitation of Oligonucleotides

F Northern Blot Analysis of Small RNA Molecules

G Additional Recipes

V Appendix 2

A References

B Safety Information

C Quality Control



PN 1554M Revision B Revision Date June 2 2008

For research use only Not for use in diagnostic procedures

Information in this document is subject to change without notice Applied Biosystems assumes no responsibil-ity for any errors that may appear in this document

Applied Biosystems disclaims all warranties with respect to this document expressed or implied including butnot limited to those of merchantability or fitness for a particular purpose In no event shall Applied Biosystemsbe liable whether in contract tort warranty or under any statute or on any other basis for special incidentalindirect punitive multiple or consequential damages in connection with or arising from this document

including but not limited to the use thereof

Literature Citation When describing a procedure for publication using this product please refer to it as themir Vanatrade Probe amp Marker Kit

If a paper that cites one of Ambionrsquos products is published in a research journal the author(s) may receive afree Ambion T-shirt by sending in the completed form at the back of this protocol along with a copy of thepaper

Warranty and Liability Applied Biosystems is committed to delivering superior product quality and perfor-mance supported by industry-leading global service and technical support teams Warranty information forthe accompanying consumable product is available at wwwambioncominfowarranty in ldquoLimited Warranty for Consumablesrdquo which is subject to the exclusions conditions exceptions and limitations set forth under

the caption ldquoEXCLUSIONS CONDITIONS EXCEPTIONS AND LIMITATIONSrdquo in the full warranty statement Please contact Applied Biosystems if you have any questions about our warranties or would likeinformation about post-warranty support

Trademarks Applied Biosystems AB (Design) Ambion BrightStar FirstChoice MAXIscript Northern-Max and ULTRAhyb are registered trademarks and Decade and mir Vana are trademarks of Applera Corpora-tion or its subsidiaries in the US andor certain other countries All other trademarks are the sole property of their respective owners

copy 2008 Ambion Inc All Rights Reserved



IA Product Description and Background Information

Introduction

I Introduction

A Product Description and Background Information

Brief product description Noncoding small RNAs such as transfer RNA (tRNA) ribosomal RN(rRNA) small nucleolar RNA (snoRNA) and small nuclear RN(snRNA) play critical roles in a variety of biological processes In tpast few years two novel classes of small RNAs microRNA (miRNand small interfering RNA (siRNA) have also emerged as powerposttranscriptional regulators of gene expression (Carrington a Ambros 2003 Pasquinelli and Ruvkun 2002) Short probes are essentto detect small RNAs for analysis of their biogenesis and function Tmir Vanatrade Probe amp Marker Kit is designed for rapid 5 end labeling aclean-up of small RNA or DNA probes The kit also contains reagento prepare small radiolabeled RNA size markers (Decadetrade Marke

150 100 90 80 70 60 50 40 30 20 and 10 nt) and single-nucotide RNA ladders (Figure 1) The purification procedure and compnents included in the kit are optimized for efficient recovery of smRNA probes and can also be used to remove unincorporated nucotides from radiolabeled probes prepared by in vitro transcription



mir Vanatrade Probe amp Marker Kit

IA Product Description and Background Information2

5 end labeling The mir Vana Probe amp Marker Kit provides optimized reagents for the5 end labeling of nucleic acids using bacteriophage T4 PolynucleotideKinase (T4 PNK) and [γ -32P]ATP (not included) T4 PNK catalyzes thetransfer of the gamma phosphate from ATP to the 5-hydroxyl of thenucleic acid molecule (thus only nucleic acids with a 5-OH group can belabeled eg RNA or DNA oligonucleotides PCR products This phos-phate transfer is commonly called a kinase or phosphorylation reaction

IMPORTANT

DNA cut with most restriction enzymes IVT RNAs or small RNAs purified

from biological samples have a 5-PO 4

and unless they are treated enzymat-

ically to remove the phosphate first are not substrates for T4 PNK

Figure 1 Probes and Markers Prepared with the mir Vanatrade

Probe amp Marker Kit

Radiolabeled probes were prepared following the instructions in section II The

32 nt RNA probe was synthesized by in vitro transcription (IVT) using themir Vana miRNA Probe Construction Kit The Purification Cartridges andreagents were used to remove unincorporated nucleotides from all the labeled

nucleic acids except the Control DNA 125 of the purified Control RNA

probe was subjected to alkaline hydrolysis as described in section IVC to pro-

duce the RNA Ladder The indicated percentage of each prep was resolved on

a 15 denaturing polyacrylamide gel The gel was exposed for 15 min

150ndash

100ndash

80ndash

70ndash

60ndash

50ndash

40ndash

ndash 32 nt

ndash 28 nt

ndash 16 nt

30ndash

20ndash

D e c a

d e trade

M a r k

e r s ( 1 0

)

C o n t r

o l D N

A p r o b

e ( 1 2 5 )

C o n t r

o l R N

A p r o b

e ( 1 2 5

)

R N A l a d

d e r ( 1

2 5 )

I V T R N

A p r o b e

( 0 5 )




Introduction

Applications Short radiolabeled probes prepared with the mir Vana Probe amp MarkKit are especially useful for the study of small RNA molecules suchrRNA snoRNA snRNA siRNA or miRNA DNA probes are typicaused for Northern Southern or primer extension experiments RNprobes are also useful in hybridization reactions because RNA-RN

hybrids are more stable than RNA-DNA or DNA-DNA duplexes addition to standard nucleic acid detection procedures which rely hybridization on a solid support (Northern Southern or dot blot) shantisense RNA probes can also be used for the sensitive detection miRNA or siRNA by solution hybridization with the mir Vana miRNDetection Kit Used with the mir Vana miRNA Isolation Kit designfor the rapid purification of representative total RNA population cotaining all of the small RNA species the mir Vana miRNA Detection Kand the mir Vana Probe amp Marker Kit are an integrated system optimizfor the study of the biogenesis and functions of siRNA and miRNA

Short RNA probes are also useful for a variety of RNA structurefun

tion experiments For more information see our web based resource a wwwambioncomtechlibresourcesstructureindexhtml

Radiolabeled RNA probes are also widely used to study protein-RNinteractions The most common applications include bandshift assaysnondenaturing gels (EMSA) in vitro crosslinking with recombinantnative proteins immunoprecipitation with antibodies specific for tprotein of interest and protein pull-down using recombinant taggfusion proteins

Control DNA Substrate The Control DNA Substrate provided with the kit can be radiolabel

to generate an antisense DNA oligonucleotide probe specific forhighly conserved portion of the 5S rRNA The probe sequence 5-TTAGCTTCCGAGATCA -3 This probe was successfully used detect 5S rRNA expression in several human mouse and rat tissues acell lines by Northern blot (see example in Figure 2 on page 4 and pcedure in section IVF on page 21) The 5S rRNA is a small RN(120 nt) constitutively expressed at a high level in all cell types Lother small RNAs 5S rRNA is not efficiently recovered using RNA islation procedures that are not specifically optimized for small RNpurification Thus 5S rRNA is an ideal loading control for experimeaimed at analyzing expression patterns of differentially expressed sm

RNAs such as miRNAs

Control RNA Substrate The Control RNA Substrate provided with the kit is an HPLC-purifiRNA oligonucleotide that can be radiolabeled to generate an antisenRNA probe specific for miR-16 miRNA (Lagos-Quintana et al 200This probe was successfully used to detect mir-16 expression in sevehuman mouse and rat tissues and cell lines by Northern blot (sexample in Figure 2 on page 4 and procedure in section IVF





page 21) The Control RNA Substrate includes 6 nt at its 3 end that arenot complementary to miR-16 This additional 6 nt sequence is cleav-able by RNases A and T1 (see sequence below) making the ControlRNA probe suitable for solution hybridization assays such as the mir-Vana miRNA Detection Kit (Figure 2) Cleavage of the noncomple-

mentary 6 nt sequence results in a clear size difference between thefull-length probe and the protected target-specific fragment after gelelectrophoresis

The target RNA sequence (miR-16 22 nt) is5-UAGCAGCACGUAAAUAUUGGCG-3

The Control RNA Substrate sequence (28 nt) is3-GAGACCAUCGUCGUGCAUUUAUAACCGC-5

Sequence not derived from the target RNA is underlined This sequence will be removed from the protected probe sequence when used in themir Vana miRNA Detection Kit

Figure 2 Examples of Small RNA Detection with the Control

Probes

32P-labeled Control probes were synthesized from the Control Substrates pro-

vided with the kit and used to detect miR-16 miRNA or 5S rRNA in

FirstChoicereg Total RNA from mouse kidney Solution hybridization assays

(top panel) were performed with 5 x 104 cpm of Control RNA probe and themir Vana miRNA Detection Kit using the recommended procedure Northern

blots (bottom panels) were hybridized with 1 x 105 cpm of Control RNA or

DNA probe per mL of hybridization solution following the procedure in sec-

tion IVF starting on page 21

P r o b

e N o

t a r g e t

0 5 micro

g

1 micro g

2 micro g

4 micro g

Control RNA Probe (miR-16)2 hr exposure

Control RNA Probe (miR-16)

24 hr exposure

Control DNA Probe (5S rRNA)1 hr exposure

Northern

Solutionhybridization



IB Reagents Provided with the Kit and Storage

Introduction



Radiolabeled ATP bull [γ ndash32P]ATP 4000ndash7000 Cimmol 10ndash150 mCimL

General laboratoryequipment and supplies

bull Constant temperature heat block (37degC and 95ndash100degC)bull Microcentrifuge

bull RNase-free 15 mL or 05 mL polypropylene microfuge tubadjustable pipettors and RNase-free tips

bull 100 ethanol (ACS grade or better)

Amount Component Storage

10 μL Decade Marker RNA below ndash70degC

40 μL T4 Polynucleotide Kinase ndash20degC

80 μL 10X Kinase Buffer ndash20degC

10 μL Control RNA Substrate ndash20degC

10 μL Control DNA Substrate ndash20degC

400 μL RNA Carrier Solution ndash20degC

1 mL Alkaline Hydrolysis Buffer ndash20degC

14 mL Gel Loading Buffer II ndash20degC

14 mL Elution Buffer ndash20degC

28 mL BindingWashing Buffer Concentrate

Add 154 mL 100 ethanol before use

4degCroom temp

200 μL 10X Cleavage Reagent room temp

40 Purification Cartridge + Tubes room temp

40 Elution Tube room temp

175 mL Nuclease-free Water any temp

Store Nuclease-free Water at ndash20degC 4degC or room temp




ID Related Products Available from Applied Biosystems 6


mir Vanatrade miRNA Probe

Construction KitPN AM1550

This kit is designed to produce short (lt100 nt) labeled RNA transcripts for

use in hybridization assays to detect small RNAs including miRNA and

siRNA The kit supplies reagents for both transcription template preparation

and RNA probe synthesis Radiolabeled probes made with the kit are ideal foruse with the mir Vana miRNA Detection Kit

mir Vanatrade miRNA Detection

KitPN AM1552

The mir Vana miRNA Detection Kit provides an extremely sensitive solutionhybridization assay capable of detecting attomole amounts of RNA In addi-

tion it can be used to simultaneously detect several small RNAs such as

miRNA and siRNA or both small RNA and long RNA species in the same

sample For a complete solution for small RNA analysis use this kit in con-

junction with the mir Vana miRNA Probe Construction Kit andor the mir-

Vana Probe amp Marker Kit

mir Vanatrade miRNA Isolation

KitPN AM1560

The mir Vana miRNA Isolation Kit (patent pending) is designed especially for

the isolation of small RNAs such as microRNA (miRNA) small interfering

RNA (siRNA) and small nuclear RNA (snRNA) from tissues and cells The

kit uses a fast and efficient glass fiber filter (GFF) based procedure to isolatetotal RNA ranging in size from kilobases down to 10-mers It also includes a

procedure to enrich the population of RNAs that are 200 bases and smaller

which enhances the sensitivity of small RNA detection by solution hybridiza-

tion and Northern blot analysis

NorthernMaxreg KitsPN AM1940 AM1946

Ambionrsquos NorthernMax Kits NorthernMax and NorthernMax-Gly com-

bine ultrasensitive reliable Northern blot protocols with unsurpassed quality

control to ensure optimal results in less time

Electrophoresis Reagentssee our web or print catalog

Ambion offers gel loading solutions agaroses acrylamide solutions powdered

gel buffer mixes nuclease-free water and RNA and DNA molecular weight

markers for electrophoresis Please see our catalog or our website

( wwwambioncom) for a complete listing as this product line is always growing

RNase-free Tubes amp Tipssee our web or print catalog

Ambion RNase-free tubes and tips are available in most commonly used sizes

and styles They are guaranteed RNase- and DNase-free See our latest catalog

or our website (wwwambioncomprodtubes) for specific information

miRNA Certified FirstChoicereg

Total RNAsee our web or print catalog

All of Ambions high quality total RNA from normal human mouse and rat

tissue is prepared by methods that quantitatively recover microRNAs The

entire line of FirstChoice Total RNAs are free of DNA and shown to be intact

by stringent quality control standards



IIA Nucleic Acid Substrate Preparation and Planning

mirVana Probe amp Marker Kit Procedure



1 Suitable nucleic acidsubstrates for T4 PNK

Use purified oligonucleotides if possible

The mir Vana Probe amp Marker Kit procedure was designed to accomodate gel-purified HPLC-purified or simply desalted DNA and RNoligonucleotides

Unpurified (desalted only) oligonucleotide preps may contain a signcant amount of smaller-than-full-length products due to poor coupliefficiency during synthesis or subsequent degradation by nuclease cotamination (to assess the quality of an oligonucleotide prep see sectiIVA starting on page 17) To obtain probes with the highest possispecific activity we recommend using purified oligonucleotides whpossible This is especially important for demanding applications th

require primarily full-length probes (eg primer extension with DNprobes or solution hybridization assays with RNA probe) Alternativedesalted oligonucleotides can be used directly in the kinase reaction afull-length radiolabeled probes can be recovered by gel purification (section IVD on page 19) This strategy is not preferred because smalproducts will compete for labeling with the full-length oligonucleotidpotentially reducing specific activity

Substrates must have a 5-OH

Any nucleic acid with a 5-OH (eg RNA or DNA oligonucleotidPCR products) can be phosphorylated by T4 PNK DNA cut with mrestriction enzymes IVT RNAs or small RNAs purified from biologi

samples have a 5-PO4 and unless they are treated enzymatically remove the phosphate first are not substrates for T4 PNK

2 Chemical inhibitors toavoid

Since ammonium ions inorganic phosphate and pyrophosphainhibit T4 PNK it is important that the nucleic acid substrate not cotain these compounds For example DNA or RNA should not be pcipitated with or dissolved in reagents containing ammonium saprior to T4 PNK reactions A monovalent cation concentratige100 mM can also compromise T4 PNK reactions because the K m ATP of T4 PNK is decreased as the monovalent cation concentrationincreased

If there is a chance that your substrate could contain any of these cheical inhibitors precipitate it to remove these compounds before usingin a mir Vana Probe amp Marker Kit end labeling reaction (precipitatiinstructions are provided in section IVE on page 20)




IIA Nucleic Acid Substrate Preparation and Planning 8

3 Calculate the molaramount of nucleic acidsubstrate and [γ ndash 32P]ATP

For optimal labeling of nucleic acid probes with T4 PNK the reactionshould contain a 2ndash5 molar excess of radiolabeled ATP over substrate(5 nucleic acid ends) Instructions for calculating the molar concentra-tion of radiolabel and substrate are shown below

[γ ndash32P]ATP

Calculate the molar concentration of radiolabeled ATP from its specificactivity (we recommend 4000ndash7000 Cimmol) and concentration (typ-ically 10ndash150 mCimL)

Oligonucleotide substrate

bull Oligonucleotides are usually supplied dehydrated We recommendresuspending them at ~100 μM (100 pmolμL) in nuclease-free water or TE buffer (10 mM Tris-HCl pH 7ndash8 01 mM EDTA)Then check the concentration of the stock solution by diluting asample 150 to 1500 in water and reading its absorbance in a spec-trophotometer at a wavelength of 260 nm Multiply the reading by the dilution factor to give A 260 then calculate the concentration withthe following equation

The extinction coefficient for individual RNA or DNA oligonucle-otides is most easily determined using a web-based calculator such asthe one at

wwwambioncomtechliboligocalc

bull Finally prepare a 05ndash10 pmolμL working solution of the nucleic

acid substrate for end labeling with this kit The two positive controloligonucleotides provided with the kit are at 5 pmolμL

molar concentration (μM or pmolμL) =specific activity (Cimmol)

concentration (mCimL) x 1000

For example 1 μL of [γ ndash32P]ATP at 10 mCimL and 6000 Cimmol contains

6000 Cimmol

10 mCimL x 1000= 167 μM or 167 pmolμL [γ ndash32P]ATP

Concentration (μM or pmolμL) =A260 x 106

(ε x L)

Whereε = extinction coefficient (L x molndash1 x cmndash1)L = path length (cm) spectrophotometers typically have a 1 cm path length



IIB 5 End Labeling Reaction



From 01ndash20 pmol of 5 ends can be efficiently end labeled p10ndash20 μL reaction Use the amount of material within the range thsuits your needs In general a labeling reaction containing 2ndash5 pmol

substrate will provide enough labeled material (after purification) many different downstream experiments including Northern blot anysis We recommend including a 2ndash5 molar excess of labeled ATP ovmolecules of substrate to obtain probe with the maximum specific actity If desired as little as a 1ndash2 fold molar excess of ATP can be usedthe reaction but we do not recommend using less than an equal moamount of ATP over substrate (see section IIA3 on page 8 for instrutions on calculating molar amounts of substrate and labeled ATP)

1 Thaw the frozen reactioncomponents at room

temp

Thaw the following reaction components and reagents at room temthen briefly vortex each before use

bull DNA or RNA substratebull [γ ndash32P]ATP (4000ndash7000 Cimmol 10ndash150 mCimL)

bull 10X Kinase Buffer

bull Nuclease-free Water

IMPORTANT

Keep the tube of T4 PNK at ndash20degC and do not vortex it

2 Assemble the reaction atroom temp

a In a microfuge tube mix the following at room temp

NOTE

The reaction can be assembled in a final volume of 20 μL if required

accommodate the substrate andor [ γ ndash32 P]ATP volume For 20 μL re

tions use 2 μL of 10 X Kinase Buffer (1X final concentration)

b Gently mix by pipetting or gentle vortexing Centrifuge brieflycollect the mixture at the bottom of the tube

3 Incubate at 37degC for 1 hr Incubate the 5 end labeling reaction for 1 hr at 37degC

Amount Component

to 10 μL Nuclease-free Water

01ndash20 pmol DNA or RNA substrate

1ndash40 pmol [γ -32P]ATP

1 μL 10X Kinase Buffer

1 μL T4 PNK (10 UμL)




IIB 5 End Labeling Reaction10

4 After the labeling reaction Storage

Store radiolabeled nucleic acids at ndash20degC or ndash80degC and keep them onice when in use Because of radiolytic decay they have a limited shelf lifemdash typically less than 1 week

No purification

For many applications (eg Northern blots) end labeled nucleic acid canbe used directly without any purification However if the T4 PNK must be inactivated (eg for subsequent enzymatic reactions) addEDTA to 1 mM and heat the reaction at 95degC for 2 min

Purification options

Below are listed common cleanup options and when they might beappropriate

bull If it is important to remove the kinase reaction reagents and the freenucleotides (eg to accurately quantitate the radiolabeled probe ormarker) purify the labeled nucleic acid immediately after step 3 fol-

lowing the procedure described in section IID starting on page 12bull If the oligonucleotide substrate did not consist of primarily

full-length material (see section IVA Gel Analysis of Oligonucleotides and Labeling Products starting on page 17) the full-length radiola-beled probes can be recovered by gel purification (see section IVDGel Purification of Probe starting on page 19)

Specific activity

The specific activity of the reaction product will depend primarily onthe specific activity of the [γ -32P]ATP Other factors such as the currentdate in relation to the reference date for the radionucleotide and the

quality of the substrate will also have some impactTo measure the specific activity of the probe reserve a 1 μL aliquot of the reaction before column or gel purification Measure and comparethe total amount of radiolabel present in the reaction mixture with thetrichloroacetic acid (TCA) precipitable counts as described in sectionIVB on page 17



IIC Preparation of Radiolabeled Decade Markers



Radiolabeled Decade Markers are prepared from the Decade MarkRNA substrate provided with the kit The RNA is first 5 end labeland the reaction is then diluted into the 10X Cleavage Reagent that ge

erates the molecular weight marker set in a 5 minute reactionThe resulting Decade Markers can be used without any purificationthey can be purified to remove unincorporated [γ ndash32P]ATP and allprecise quantitation by scintillation counting This is advantageous some experiments because it provides a way to determine the volumemarker to load on a gel for a given exposure time The purificatishould be done immediately after the labeling reaction and the cleavareaction is then carried out using purified 5 end labeled transcript

1 Assemble the 5 endlabeling reaction

2 Incubate 1 hr at 37degC Incubate the 5 end labeling reaction for 1 hr at 37degC

bull If you do not plan to remove unincorporated [γ ndash32P]ATP proce

to step 3 below

bull To make purified Decade Markers complete the column purifition described in section IID starting on page 12 and then proceto step 3 below

3 Add 10X CleavageReagent

Unpurified labeling reactions

Add 8 μL of Nuclease-free Water and 2 μL of 10X Cleavage Reagent

Purified labeling reactions

After completing step IID6 on page 13 of the column purification

bull Measure the exact volume of eluted material with a pipet

bull Add 110 volume of 10X Cleavage Reagent

4 Mix thoroughly Gently mix by pipetting or slow vortexing Centrifuge briefly to collthe mixture at the bottom of the tube

5 Incubate 5 min at roomtemp

Incubate the cleavage reaction for 5 min at room temp

Amount Component


1 μL Decade Marker (~1 pmol)

ge1 pmol [γ ndash32P]ATP

For example 1 μL of 6000 Cimmol [γ ndash32P]ATP at aconcentration of 10 mCimL provides ~167 pmol






IID Column Purification to Remove Free Nucleotides 12

6 Add an equal volume GelLoading Buffer IIincubate 3 min at 95degC

Stop the cleavage reaction by adding an equal volume of Gel LoadingBuffer II and heating at 95degC for 3 min The Decade Markers are now ready for gel electrophoresis

7 Storage and usage

instructions

bull Store the Decade Markers at ndash20degC for up to 10 days

bull To use the Decade Markers thaw the frozen solution remove anappropriate volume and heat at 95degC for 3 min before loading onthe gel

bull The Decade Markers can be used in any denaturing acrylamide gelhowever we find that using a denaturing (7 M urea)10ndash20 acrylamide gel provides the best resolution of theten Decade Marker fragments An 11th band (150 nt) is also visibleits intensity will be about 2ndash5-fold higher than the rest of the mark-ers The appropriate volume of Decade Markers to load on a gel willdepend on the size of the wells the age of the radioisotope and theexposure time


The purification procedure was designed to quickly remove unincorpo-rated radiolabeled nucleotides and other labeling reagents from endlabeled RNA It is convenient because only a single tube is used and aminimal amount of radioactive waste is produced The procedure canalso be used to efficiently purify radiolabeled RNA molecules preparedby in vitro transcription for example with the mir Vana miRNA ProbeConstruction Kit or the MAXIscriptreg Kit An RNA Carrier Solution ismixed with the labeled RNA for optimal recovery (up to 100) of small

amounts of very short RNAsThe protocol can also be used to clean up DNA labeling reactionsHowever the recovery will not be as efficient as for RNA (typically lessthan 50 of the RNA recovery) The RNA Carrier Solution must not beused to purify radiolabeled DNA oligonucleotides as it will furtherreduce their recovery Start at step 2 for labeled DNA

Add 154 mL of 100 ethanol to the BindingWashing BufferConcentrate before use

Use ACS grade or better 100 ethanol to make a working solution of the BindingWashing Buffer and mix well

1 Add 10 μL of RNA CarrierSolution to RNA labelingreactions

Start here for purification of RNA (Go to step 2 for purification of DNA)

Add 10 μL of RNA Carrier Solution to

bull RNA 5 end labeling reactions immediately after the kinase reaction(step IIB3)

bull Decade Marker 5 end labeling reactions immediately after thekinase reaction (step IIC2)



IID Column Purification to Remove Free Nucleotides


bull In vitro transcription reactions immediately after the DNase treatm(eg step IIC5 in the mir Vana Probe Construction Kit Protocol)

NOTE

The RNA Carrier Solution contains sheared yeast RNA (1 mgmL) that

be recovered with the purified RNA at the end of the procedure Ye

RNA is often used as a nonspecific competitor in molecular biology p

cedures and will be compatible with most downstream applicatio

However omit the RNA Carrier Solution if yeast RNA is expected to int

fere with subsequent enzymatic reactions or functional assays

2 Add 350 μLBindingWashing Bufferwith ethanol

Start here for purification of DNA

IMPORTANT

Add 154 mL of 100 ethanol ACS grade or better to the BindingWash

Buffer Concentrate before use

Add 350 μL BindingWashing Buffer to each sample and mix th

oughly by pipetting a few times or by inverting the tube several time

3 Draw the mixturethrough a PurificationCartridge

a Apply the mixture to a Purification Cartridge pre-assembled in onethe 2 mL Collection Tubes provided

b Centrifuge 15ndash30 sec at ~10000 x g

4 Wash with 300 μLBindingWashing Buffer

a Apply 300 μL BindingWashing Buffer to the same PurificatiCartridge and Collection Tube (the capacity of the Collection Tubis 700 μL)

b Centrifuge at least 1 min at ~10000 x g

5 Elute nucleic acid with10ndash40 μL of ~95degC ElutionBuffer

a Transfer the Purification Cartridge into a fresh Elution Tube

b Apply preheated (~95ndash100degC) Elution Buffer to the PurificatiCartridgeThe exact volume of Elution Buffer is not critical For optimal recoery of nucleic acid we recommend eluting with two sequential etions of 20 μL Elution Buffer each

c Recover the nucleic acid by centrifugation at ~10000 x g for ~30 s

6 Elute with a second10ndash20 μL aliquot of ~95degCElution Buffer

Apply a second aliquot of hot Elution Buffer to the filter and respin ~30 sec This second elution is collected in the same tube as the first eluti

NOTE

Elution Buffer is nuclease-free

10 mM Tris pH 7 01 mM EDTA





7 After the purification To make purified Decade Markers

To continue preparation of labeled Decade Markers proceed tostep C3 Purified labeling reactions on page 11

Storage

Store radiolabeled nucleic acids at ndash20degC or ndash80degC and keep them on

ice when in use Because of radiolytic decay they have a limited shelf lifemdash typically less than 1 week

Determine cpm per μL

Scintillation count 1 μL of the purified material to determine theamount of radioactivity present (cpmμL)



IIIA Using the Control Substrates

Troubleshooting

III Troubleshooting


Description There are Control RNA and Control DNA Substrates (5 μM) for 5 elabeling reactions provided with the mir Vana Probe amp Marker Kit TControl RNA Substrate is a 28 nt RNA oligonucleotide that is compmentary to miR-16 miRNA Once labeled it can be used as a probe fboth solution hybridization with the mir Vana miRNA Detection Kand for Northern blotting (procedure in section IVF) The ContDNA Substrate is a 16 nt DNA oligonucleotide that is complementto 5S rRNA and can be used for Northern blotting

Instructions To do the positive control reaction use 1 μL or less of either the Cotrol RNA or DNA Substrate (2ndash5 pmol) in a 5 end labeling reacti with at least 5 pmol of fresh radiolabeled ATP (eg 3 μL of [γ ndash32P]ATat 6000 Cimmol and 10 mCimL) Follow the instructions in sectiIIB on page 9 If desired the labeled Control RNA can be purified flowing the instructions in section IID on page 12

Expected result Positive control reactions using either the RNA or DNA Control Sustrate should yield labeled nucleic acid with a specific activity of at le106 cpmpmol (see section IVB to determine percentage incorporation and specific activity)Representative examples of radiobeled probes and their usage in a solution hybridization assay or inNorthern blot are shown in Figure 1 on page 2 and Figure 2 on page


1 Try the positive control The first step in troubleshooting poor labeling is to check that the components are functioning properly by using the Decade Marker RNor one of the Control Substrates in a 5 end labeling reaction described in section IIIA above If your experiments involve labeliRNA we recommend doing the control reaction with the Control RNSubstrate If you will be labeling DNA the Control DNA Substrateprobably a more pertinent control




IIIB Troubleshooting Poor Labeling 16

2 The positive controlworks but my samplesdonrsquot label well

a There is an inhibitor in the nucleic acid substrate

As discussed in section IIA2 on page 7 the quality of the substratecan be a significant factor in the performance of the kit Consider whether your preparation contains any of the following reactioninhibitors and if necessary clean up the nucleic acid you want to

labelbull residual ammonium salts

bull inorganic phosphate andor pyrophosphate

bull monovalent cation concentration 100 mM

b Only nucleic acids with a 5-OH are a substrate for T4 PNK

Nucleic acids such as PCR products and chemically synthesizedRNA or DNA oligonucleotides typically have a 5-OH (hydroxylgroup) DNA from restriction enzyme digestion nucleic acids puri-fied from biological samples and in vitro transcribed RNAs typically do not have a 5-OH and thus are not substrates for T4 PNK

c Check the concentration of nucleic acids and [γ -32

P]ATPFor optimal labeling of nucleic acid probes with T4 PNK a2ndash5 molar excess of ATP over 5 nucleic acid ends is recommendedDetermine the exact concentration of each substrate following theinstructions in section IIA3

d 5 end of oligonucleotide is inaccessible because of tertiary

structure

The 5 end of an oligonucleotide may be occluded by its tertiary structure reducing the efficiency of the kinase reaction To work around this problem heat the sample to 90degC for 5 min then placeit immediately on ice just before the reaction To start the reaction

add the denatured oligonucleotide directly to a mixture of the rest of the reagents



IVA Gel Analysis of Oligonucleotides and Labeling Products

Additional Procedures



The best way to quickly analyze RNA or DNA oligonucleotides eith

before or after a labeling reaction is to electrophorese a sample ondenaturing 12 or 15 polyacrylamide gel (see section IVGpage 22 for gel recipes) The procedure described here can be used check the quality of chemically synthesized DNA or RNA oligonucotides and to analyze radiolabeled DNA or RNA oligonucleotidbefore or after column purification (to verify their integrity)

1 Mix a sample of the oligonucleotide with Gel Loading Buffer II ifinal volume of 5ndash10 μLbull Use 1ndash5 of the labeling reaction for radiolabeled probes

bull Use 50 to 200 pmol for oligonucleotides (eg 05ndash2 μL o100 μM stock)

2 Heat the mixture for 3 min at 95ndash100degC

3 Load the sample on a denaturing 12 or 15 polyacrylamide and electrophorese at 20ndash25 mA

4 Stop electrophoresis when the bromophenol blue dye front hmigrated to the bottom of the gel

5 Expose to X-ray film for one to several minutes for radiolabeled ogonucleotides or stain the gel with ethidium bromide as describbelow for unlabeled oligonucleotidesbull Soak the gel for 5 min in a 05ndash1 μgmL solution of ethidiu

bromide in 1X TBE

bull Wash the gel for 2ndash5 min in 1X TBE

bull Visualize oligonucleotides using a UV transilluminator


The specific activity of a labeled nucleic acid reflects the efficiency of tkinase reaction and provides a useful number for subsequent expements Specific activity is simply cpmpmol calculated before purifition Doing the trichloroacetic acid (TCA) precipitation ascintillation counting described below will also tell you how macpmμL are in the tube and the percentage of the radiolabel that w

incorporated into TCA precipitable material

You will not be able to determine the specific activity of radiolabenucleic acid that has been purified away from unincorporated nucotides You can however assay a small aliquot of the labeled nuclacid after purification to determine its cpmμL




IVB Determining Percent Incorporation and Specific Activity 18

1 Dilute the labeled nucleicacid into carrier nucleicacid

a Mix 1 μL of the labeling reaction or purified reaction with 9 μLnuclease-free water in a fresh microfuge tube (110 dilution)

b Dispense 198 μL carrier DNA or RNA (1 mgmL) into anuclease-free 15 mL microfuge tube (Ambion Sheared SalmonSperm DNA PN AM9680 can be used for this) Add 2 μL of the

diluted mir Vana Probe amp Marker Kit reaction to the carrier DNA orRNA and mix thoroughly

2 Measure the total amountof radiolabel in thereaction mixture

Transfer 100 μL of the mixture from step 1b to aqueous scintillationcocktail and count in a scintillation counter This will measure the totalamount of radiolabel present in the reaction mixture (unincorporatedand incorporated counts)

3 Measure TCA-precipitablecounts

a Transfer another 100 μL of the mixture from step 1b to a12 x 75 mm glass tube and add 2 mL of cold 10 TCA Mixthoroughly and place on ice for 10 min This will precipitate nucleic

acids but not free nucleotidesb Collect the precipitate via vacuum filtration through a Whatman

GFC glass fiber filter (or its equivalent)

c Rinse the tube twice with 1 mL of 10 TCA and then rinse once with 3ndash5 mL of 95 ethanol Pass each of the rinses through theGFC filter

d Place the filter in a scintillation vial add aqueous scintillationcocktail and count in a scintillation counter The number will reflectradiolabel that was incorporated

4 Calculate incorporationand specific activity

Divide the cpm in step 3d by the cpm in step 2 to determine the frac-tion of label incorporated (multiply by 100 for percent incorporation)

To determine the specific activity of the labeled nucleic acid calculatethe total cpm incorporated in the reaction and divide it by the pmolamount of nucleic acid substrate in the reaction

Total cpm incorporated = cpm from step 3d x dilution factor (10) x kinase reaction volume (10 or 20)

Specific activity =total cpm incorporated

pmol substrate in the reaction



IVC Alkaline Hydrolysis of RNA Probes



Purified 5 end labeled RNA (unincorporated nucleotides removed) cabe rapidly hydrolyzed to generate a gel electrophoresis ldquoladderrdquo of 5 enlabeled RNA fragments This procedure suggests using 3 differe

hydrolysis times After the experiment select the ladder that providthe best distribution of nucleic acids over the size range best suited your experiments At least one of the time points should yield a signifcant amount of full-length RNA and a single base ladder of RNA banbelow

1 In an RNase-free microfuge tube mix 5 μL of purified 5 end labelRNA with 10 μL Alkaline Hydrolysis Buffer

NOTE

The total amount of probe is not critical If desired the probe can b

diluted with the provided Nuclease-free Water to a final volume of 5 μL

2 Split the mixture into 3 tubes containing 5 μL each

3 Place the tubes in a 95degC incubator

4 Stop the reactions at three different time points by adding 10 μL Gel Loading Buffer IIThe optimal incubation time must be determined empirically fexample by testing 5 10 and 15 min With the Control RNA pro we typically stop the reactions at 6 8 and 10 min

5 Separate the hydrolysis products on a denaturing gel and visualithem by autoradiographyLoad 3ndash5 μL of each reaction on a denaturing 12ndash15 polyacrylami

gel (recipe in section IVG on page 22) according to the size ranappropriate for your experiment It is a good idea to run an untreatcontrol RNA sample next to the RNA ladder for comparison


Specific applications such as ribonuclease protection assays S1 nucleaassays and DNA primer extension require full-length probes If the ogonucleotide substrate in the kinase reaction was not comprised primrily of full-length material we recommend gel purifying the probe separate full-length oligonucleotides from shorter products as well

from unincorporated nucleotides This procedure can be done directafter the labeling reaction or after column purification

1 Separate products on anacrylamide gel

Add an equal volume of Gel Loading Buffer II to the sample and hefor 3 min at 95ndash100degC Load the products on a denaturing polyacrylmide gel and electrophorese the gel at ~10ndash25 mA until the bromophnol blue reaches the bottom of the gel (See section IVG on page 22 facrylamide concentration guidelines and gel recipes) An entire labelin




IVE Precipitation of Oligonucleotides 20

reaction (10 μL plus 10 μL Gel Loading Buffer II) can be loaded in asingle standard 10 x 5 x 075 mm well For larger volumes use a ldquopre-parative scalerdquo comb with wide teeth that will form large capacity wells

2 Excise the gel fragment

containing the full-lengthnucleic acid

After electrophoresis remove one glass plate from the gel cover the gel

with plastic wrap and expose it to X-ray film for 30 sec to several min-utes the exposure time will depend on the specific activity of the probeThe goal is to get an autoradiograph with a faint or ldquogreyrdquo signal so thata small discrete gel fragment can be excised Glow-in-the-dark stickersare the easiest way to orient the film with the gel to cut out the bandOnce exposed develop the film and identify the full-length probe it isusually the most slowly migrating most intense band on the autoradio-graph Now align the exposed X-ray film with the gel and cut out thearea of the gel that contains the full-length labeled probe with a razorblade or a scalpel and transfer it to an RNase-free microfuge tubeRemove the smallest possible fragment of gel that contains the full

length probe

3 Elute nucleic acid fromacrylamide gel slices

To elute the full-length probe add 100ndash150 μL probe elution buffer(05 M ammonium acetate 1 mM EDTA 02 SDS) to the gel sliceand incubate at 37degC for 30 min Transfer the buffer which containsthe eluted probe to a clean microfuge tube and repeat with 50ndash100 μLof probe elution buffer Pool the two elution fractions and determinethe cpmμL of the recovered probe by scintillation counting using1ndash2 μL of the eluted material

Since longer probes elute more slowly from the gel we recommendincreasing the elution time for probes longer than 40 nt to at least 1 hr

For convenience or to maximize recovery from the gel incubation canbe carried out overnight with ~200 μL or more of Probe Elution BufferThe Probe Elution Buffer contains EDTA and SDS which will inacti-vate low levels of nuclease contamination

If your application requires it salt and SDS can be removed by ethanolprecipitation (see section IVE below)


1 Add 110th volume 3M sodium acetate and ge3 volumes100 ethanol and mix well

2 Chill at or below ndash20degC for ge30 min

3 Spin at top speed in a microfuge for ge20 min and discard the super-natant

4 Rinse the pellet with cold 75 ethanol and recentrifuge at topspeed for ge5 min

5 Air dry the pellet



IVF Northern Blot Analysis of Small RNA Molecules


2


Northern blots can provide qualitative and quantitative data for retively abundant small RNAs For less abundant RNAs such as specimiRNAs we recommend using the more sensitive mir Vana miRN

Detection Kit procedure or analyzing RNA fractions that are enrichin small RNAs species with the mir Vana miRNA Isolation Kit

Specific small RNAs can be detected on Northern blots using eithantisense RNA probes prepared by in vitro transcription or 5 elabeled antisense RNA or DNA probes Below we provide a proceduusing Ambion optimized NorthernMaxreg reagents for RNA probes aa procedure adapted from Patterson and Guthrie for DNA prob(Patterson 1987)

1 Run RNA sample on anacrylamide gel

Run 1ndash50 μg of total RNA or an RNA fraction enriched in small RNon a denaturing 15 polyacrylamide gel (see section IVG for recip

Stop electrophoresis when the bromophenol blue dye front hmigrated to the bottom of the gel Stain the gel with ethidium bromidand examine it on a transiluminator to assess the quality of the sampand to make sure that there is good separation of the RNA

2 Transfer RNA tohybridization membrane

After staining transfer the RNA to a nylon membrane by electroblting (Ambion offers BrightStarreg-Plus Nylon membraPN AM10100) This procedure can be performed in a semi-dry appratus using a stack of three sheets of blotting paper soaked in 025X TBplaced above and below the gelmembrane For 075 mm gels transat 200 mA for at least 02 A-hr (A x hr) Extending this time does nresult in loss of sample After blotting keep the membrane damp aUV crosslink the RNA to the membranes using a commercial crosslining device (120 mJ burst over 30 sec)

3

3a RNA probes a Prehybridize membrane in ULTRAhybreg-Oligo Hybridization Buf(Ambion PN AM8663) for at least 1 hr at 65degC

b Add 1ndash5 x 105 cpm RNA probe per mL of ULTRAhyb-Oligo

c Hybridize 8ndash24 hr at 42degC

d Wash 3 times for 5 min each at room temp with NorthernMax L

Stringency Wash Buffer (PN AM8673)e Wash 15 min at 42degC with NorthernMax Low Stringency Wa

Buffer




IVG Additional Recipes 22

3b DNA probes a Prehybridize membrane in prehybridization solution (recipe insection IVG) for at least 1 hr at 65degC

b Discard the prehybridization solution

c Add 1ndash5 x 105 cpm DNA probe per mL of hybridization solution(recipe in section IVG)

d Hybridize 8ndash24 hr at room temp

e Wash 3 times for 5 min each at room temp with Northern washsolution (recipe in section IVG)

f Wash 15 min at 42degC with Northern wash solution

4 Expose the membrane After the final wash wrap the blot in plastic wrap and expose to X-ray film or a phosphorimager screen according to the manufacturer instruc-tions The latter method allows quantification of the amount of signalpresent in each band

5 Detecting larger RNAspecies

To look for larger RNAs (eg mRNAs which are not resolved on anacrylamide gel) in the same RNA samples you would have to make aNorthern blot using an agarose gel system (Ambion NorthernMaxreg KitsPN AM1940 AM1946 are ideal for this)


1 Recommendedpolyacrylamideconcentration

2 10X TBE TBE is generally used at 1X final concentration for preparing gelsandor for gel running buffer

IMPORTANT

Do not treat TBE with diethylpyrocarbonate (DEPC)

Dissolve with stirring in about 850 mL nuclease-free water Adjust thefinal volume to 1 L

NucleicAcid Size

PolyacrylamideConcentration

Xylene CyanolPosition

Bromophenol BluePosition

gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt

Concentration Component for 1 L

09 M Tris base 109 g

09 M Boric Acid 55 g

20 mM 05 M EDTA solution 40 mL



IVG Additional Recipes


2

Alternatively Ambion offers nuclease-free solutions of 10X TB(PN AM9863 AM9865) and ready-to-resuspend powdered 10X TBpackets (PN AM9864) Both are made from of ultrapure molecubiology grade reagents

3 Denaturing acrylamidegel mixes

Use the following instructions to prepare 15 mL of gel mix with tindicated percentage of acrylamide and 8 M urea 15 mL is enough solution for one 13 x 15 cm x 075 mm gel Ambion offers reagents acrylamide gel preparation see our latest catalog or our web site for spcific information

Mix briefly after adding the last 2 ingredients and pour gel immeately

4 50X Denhardtrsquos Solution

5 20X SSC

Alternatively Ambion offers ready-to-use 20X SSC (PN AM976 AM9765 AM9770) or ready-to-resuspend powder (PN AM9764)

Amount

12 gel 15 gel Component

72 g 72 g Urea

15 mL 15 mL 10X TBE

45 mL 56 mL 40 acrylamide (acrylamidebis acrylamide = 191

to 15 mL to 15 mL Nuclease-free water

Stir to mix then add

75 μL 75 μL 10 ammonium persulfate

15 μL 15 μL TEMED

Amount Component

10 g Ficoll 40010 g bovine serum albumin

10 g polyvinylpyrrolidone

to 1L nuclease-free water

Amount Component

1753 g NaCl

882 g sodium citrate

800 mL nuclease-free water

pH to 70 with HClto 1 L nuclease-free water





6 Northern blotprehybridization solutionfor DNA probes

7 Northern blothybridization solution forDNA probes

Add 1ndash5 x 106 cpmmL 5 end labeled antisense probe Filter solution with 045 μm pore filter before use to remove background flecks

8 Northern wash solution

for DNA probes

Amount Component

6X SSC

10X Denhardts solution

02 SDS

Amount Component

6X SSC


02 SDS

Amount Component6X SSC

02 SDS



VA References

Appendix

2

V Appendix

A References

Carrington JC and Ambros V (2003) Role of microRNAs in plant and animal development Scien

301(5631) 336ndash8

Lagos-Quintana M Rauhut R Lendeckel W and Tuschl T (2001) Identification of novel genes coding fsmall expressed RNAs Science 294(5543) 853ndash8

Pasquinelli AE and Ruvkun G (2002) Control of developmental timing by micrornas and their targets AnRev Cell Dev Biol 18 495ndash513

Patterson B and Guthrie C (1987) An essential yeast snRNA with a U5-like domain is required for splicingvivo Cell 49(5) 613ndash24


The MSDS for any chemical supplied by Applied Biosystems Ambion is available to you free 24 hours a day

IMPORTANT

For the MSDSs of chemicals not distributed by Applied Biosystems

Ambion contact the chemical manufacturer

To obtain Material SafetyData Sheets

bull Material Safety Data Sheets (MSDSs) can be printed or downloadfrom product-specific links on our website at the following addres wwwambioncomtechlibmsds

bull Alternatively e-mail your request toMSDS_Inquiry_CCRMappliedbiosystemscom Specify the calog or part number(s) of the product(s) and we will e-mail the asciated MSDSs unless you specify a preference for fax delivery

bull For customers without access to the internet or fax our technical svice department can fulfill MSDS requests placed by telephone postal mail (Requests for postal delivery require 1ndash2 weeks for prcessing)

Chemical safety guidelines To minimize the hazards of chemicals

bull Read and understand the Material Safety Data Sheets (MSDS) p

vided by the chemical manufacturer before you store handle work with any chemicals or hazardous materials

bull Minimize contact with chemicals Wear appropriate personal prottive equipment when handling chemicals (for example safety glassgloves or protective clothing) For additional safety guidelines cosult the MSDS




VC Quality Control 26

bull Minimize the inhalation of chemicals Do not leave chemical con-tainers open Use only with adequate ventilation (for example fumehood) For additional safety guidelines consult the MSDS

bull Check regularly for chemical leaks or spills If a leak or spill occursfollow the manufacturerrsquos cleanup procedures as recommended on

the MSDSbull Comply with all local stateprovincial or national laws and regula-

tions related to chemical storage handling and disposal

C Quality Control

Functional Analysis The Control RNA and DNA Substrates are labeled following the proce-dure outlined in this Protocol The resulting probes are analyzed by PAGE

Nuclease testing Relevant kit components are tested in the following nuclease assays

RNase activity

Meets or exceeds specification when a sample is incubated with labeledRNA and analyzed by PAGE

Nonspecific endonuclease activity

Meets or exceeds specification when a sample is incubated with super-coiled plasmid DNA and analyzed by agarose gel electrophoresis

Exonuclease activity

Meets or exceeds specification when a sample is incubated with labeleddouble-stranded DNA followed by PAGE analysis

Protease testing Meets or exceeds specification when a sample is incubated with proteasesubstrate and analyzed by fluorescence



PN 1554M Revision B Revision Date June 2 2008

For research use only Not for use in diagnostic procedures

Information in this document is subject to change without notice Applied Biosystems assumes no responsibil-ity for any errors that may appear in this document

Applied Biosystems disclaims all warranties with respect to this document expressed or implied including butnot limited to those of merchantability or fitness for a particular purpose In no event shall Applied Biosystemsbe liable whether in contract tort warranty or under any statute or on any other basis for special incidentalindirect punitive multiple or consequential damages in connection with or arising from this document

including but not limited to the use thereof

Literature Citation When describing a procedure for publication using this product please refer to it as themir Vanatrade Probe amp Marker Kit

If a paper that cites one of Ambionrsquos products is published in a research journal the author(s) may receive afree Ambion T-shirt by sending in the completed form at the back of this protocol along with a copy of thepaper

Warranty and Liability Applied Biosystems is committed to delivering superior product quality and perfor-mance supported by industry-leading global service and technical support teams Warranty information forthe accompanying consumable product is available at wwwambioncominfowarranty in ldquoLimited Warranty for Consumablesrdquo which is subject to the exclusions conditions exceptions and limitations set forth under

the caption ldquoEXCLUSIONS CONDITIONS EXCEPTIONS AND LIMITATIONSrdquo in the full warranty statement Please contact Applied Biosystems if you have any questions about our warranties or would likeinformation about post-warranty support

Trademarks Applied Biosystems AB (Design) Ambion BrightStar FirstChoice MAXIscript Northern-Max and ULTRAhyb are registered trademarks and Decade and mir Vana are trademarks of Applera Corpora-tion or its subsidiaries in the US andor certain other countries All other trademarks are the sole property of their respective owners

copy 2008 Ambion Inc All Rights Reserved




Introduction

I Introduction









IMPORTANT













150ndash

100ndash

80ndash

70ndash

60ndash

50ndash

40ndash

ndash 32 nt

ndash 28 nt

ndash 16 nt

30ndash

20ndash

D e c a

d e trade

M a r k

e r s ( 1 0

)

C o n t r

o l D N

A p r o b

e ( 1 2 5 )

C o n t r

o l R N

A p r o b

e ( 1 2 5

)

R N A l a d

d e r ( 1

2 5 )

I V T R N

A p r o b e

( 0 5 )




Introduction








RNAs such as miRNAs












Probes








P r o b

e N o

t a r g e t

0 5 micro

g

1 micro g

2 micro g

4 micro g



24 hr exposure


Northern





Introduction




















4degCroom temp


















KitPN AM1552








KitPN AM1560


















































[γ ndash32P]ATP











6000 Cimmol



(ε x L)










temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity










Introduction

I Introduction









IMPORTANT













150ndash

100ndash

80ndash

70ndash

60ndash

50ndash

40ndash

ndash 32 nt

ndash 28 nt

ndash 16 nt

30ndash

20ndash

D e c a

d e trade

M a r k

e r s ( 1 0

)

C o n t r

o l D N

A p r o b

e ( 1 2 5 )

C o n t r

o l R N

A p r o b

e ( 1 2 5

)

R N A l a d

d e r ( 1

2 5 )

I V T R N

A p r o b e

( 0 5 )




Introduction








RNAs such as miRNAs












Probes








P r o b

e N o

t a r g e t

0 5 micro

g

1 micro g

2 micro g

4 micro g



24 hr exposure


Northern





Introduction




















4degCroom temp


















KitPN AM1552








KitPN AM1560


















































[γ ndash32P]ATP











6000 Cimmol



(ε x L)










temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity












IMPORTANT













150ndash

100ndash

80ndash

70ndash

60ndash

50ndash

40ndash

ndash 32 nt

ndash 28 nt

ndash 16 nt

30ndash

20ndash

D e c a

d e trade

M a r k

e r s ( 1 0

)

C o n t r

o l D N

A p r o b

e ( 1 2 5 )

C o n t r

o l R N

A p r o b

e ( 1 2 5

)

R N A l a d

d e r ( 1

2 5 )

I V T R N

A p r o b e

( 0 5 )




Introduction








RNAs such as miRNAs












Probes








P r o b

e N o

t a r g e t

0 5 micro

g

1 micro g

2 micro g

4 micro g



24 hr exposure


Northern





Introduction




















4degCroom temp


















KitPN AM1552








KitPN AM1560


















































[γ ndash32P]ATP











6000 Cimmol



(ε x L)










temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity










Introduction








RNAs such as miRNAs












Probes








P r o b

e N o

t a r g e t

0 5 micro

g

1 micro g

2 micro g

4 micro g



24 hr exposure


Northern





Introduction




















4degCroom temp


















KitPN AM1552








KitPN AM1560


















































[γ ndash32P]ATP











6000 Cimmol



(ε x L)










temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity

















Probes








P r o b

e N o

t a r g e t

0 5 micro

g

1 micro g

2 micro g

4 micro g



24 hr exposure


Northern





Introduction




















4degCroom temp


















KitPN AM1552








KitPN AM1560


















































[γ ndash32P]ATP











6000 Cimmol



(ε x L)










temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity










Introduction




















4degCroom temp


















KitPN AM1552








KitPN AM1560


















































[γ ndash32P]ATP











6000 Cimmol



(ε x L)










temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity



















KitPN AM1552








KitPN AM1560


















































[γ ndash32P]ATP











6000 Cimmol



(ε x L)










temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity






























[γ ndash32P]ATP











6000 Cimmol



(ε x L)










temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity















temp





IMPORTANT




NOTE






Amount Component












No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity













No purification







Specific activity













to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity

















to step 3 below












Amount Component













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity













7 Storage and usage

instructions



















NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity












NOTE









IMPORTANT

















NOTE









Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity













Storage








Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity










Troubleshooting

III Troubleshooting






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity






















structure

















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity





















bromide in 1X TBE





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity





































NOTE






















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity
















length probe
















2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity











2










3






Buffer

















IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity























IMPORTANT



NucleicAcid Size




gt30 nt 12 ~40 nt ~15 nt

lt30 nt 15 ~30 nt ~10 nt









2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity











2






5 20X SSC


Amount


72 g 72 g Urea

15 mL 15 mL 10X TBE





15 μL 15 μL TEMED

Amount Component




Amount Component

1753 g NaCl












for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity















for DNA probes

Amount Component

6X SSC


02 SDS

Amount Component

6X SSC


02 SDS


02 SDS



VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity









VA References

Appendix

2

V Appendix

A References


301(5631) 336ndash8






IMPORTANT



















C Quality Control



RNase activity















C Quality Control



RNase activity







mirvana probe and marker kit

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