rna sample preparation and analysis tools from ambion...

1Life Technologies™ Proprietary |

RNA Sample preparation and analysis tools from Ambion and Life Technologies


Agenda

RNA introductionRNA sample preparation microRNASpecial Samples− Blood− Single Cells− FFPE− ExosomesRNA-Seq


Tissue Lysis Releases Nucleases


Working with RNAKeep an “RNAase‐free zone”− set aside an area that is not used for biological samples or enzyme work

− prescriptive steps: clean the surface with RNase ZAP®

− proscriptive remediations: clean all pipet barrels with RNase ZAP®, replace reagents , precious reagents can be checked with RNaseAlert®


Working with RNAStart with Certified RNase‐free Reagents and Plastics

RNase‐free Water and other common reagents

RNase‐free Tubes and Tips


RNAlater®


RNA Purification Products

Reagents

Columns

Plates

Beads

BrandRNA Type

TRIzol®TRIzol® LS

PureLink™mirVana™

PureLink™mRNA Catcher™

MagMax™RiboMinus™

Total RNA

Total RNAmicroRNA

Total RNAmRNA

mRNATranscriptome


Trizol®

The most trusted and referenced reagent for preparing high quality and intact RNA− Unparalled lysis capability

− Flexible formulations for difficult samples (ietissues, cells, serum, viruses, and bacteria)

− Scalable

− Single step, monophasic solutions (phenol and guanidine isothiocyanate)

− Low gDNA contamination


Purelink™ RNA Mini‐ High Yield

Simplified total RNA purification from cells and tissues using spin columns− Incl. animal, plant, yeast,

bacterial, or blood − Purify RNA in less than 20

minutes− No hazardous or toxic

chemical disposal (no phenol)

− Scalable RNA purification up to 1,000 μg of purified RNA from a single prep


• Combines best extraction with easy to use format; high yield

• Improved purity

• Recommended protocol for Microarray

TRIzol® Plus – Best of both worlds

+

11Life Technologies™ Proprietary | 11

Magnetic bead - MagMax™

Magnetic bead based purification with flexible formats

Manual single tube or 96 well formats

Automated 24 or 96 well formatsIncreased productivity from the significant time savings vs column based purification

process 96 lysates in ~15min

Simple protocol

Specialized protocols for high RNA yield and purity from diverse sample types:

Cell free viral / biological samples, swabs, blood, plants, feces, environmental samples


TaqMan® Gene Expression Cells-to-CT™ Kit

Complete Optimized Workflow-includes sample prep, DNase, reverse transcription reagents and TaqMan® Gene Expression Master Mix for convenient real-time PCR studies

Compatibility-Components were co-developed to provide the ultimate in sensitivity

Simple and Fast-Formulated master mixes mean only 6 components for entire workflow. Sample preparation can be completed in 2 steps in less than 10 minutes, and is easily

automatable.


3 Steps

1. Lysis at Room Temp

2. Reverse Transcription

3. Real-time PCR

Sample Prep:

All at room temperature

Genomic DNA removed at same time cells are lysed

Can be done directly in culture plates

No centrifugations

No organics

No filter to clog

Cells-to-CT™ workflow – 10 to 100,000 cells


Purified RNA

Cells-to-CT™Kit

Cells-to-CT™ Kit

As the sample input is decreased, the percentage loss increases during purification. So at lower cell inputs there is quite a difference compared to purified RNA.

Equal or better performance and sensitivity compared to purified RNA


•TaqMan ® Fast Cells-to-CT™ Kit lysates and purified RNA from 4000 cells were prepared and evaluated with 163 TaqMan® Gene Expression Assays.

•20% of RT reaction was lysate/RNA.

•20% of the qPCR was RT.

•Similar data seen with miRNA

Equivalent mRNA profiles from Cells-to-CT™ and pure RNA with 163 TaqMan® Assays


MicroRNAs: Macro Significance

MicroRNAs (miRNAs) are short 18-25 nt RNA molecules, found in abundance in plants and animals

miRNAs are post-transcriptional regulators that bind to complementary sequences on target mRNAs, resulting in translational repression and gene silencing

the human genome encodes about 2000 miRNAs, which may target and regulate about 50% of mammalian genes

each miRNA typically regulates multiple mRNAs, and many mRNAs are regulated by several miRNAs.


MicroRNA Purification with mirVana™ Kits

mirVana™ Kits enable isolation of total RNA while retaining the small RNA fraction - including microRNAs down to 10-mers.

Ideal for performing RNA-seq, microarrays, TaqMan®/SYBR qPCR analysis

Non-Coding RNA

Easy workflow: Sample to Purified RNA in 30 Minutes:


TaqMan® MicroRNA Cells-to-CT™ KitFast, simple, and convenient—Prepare samples in ~10 minutes; eliminate tedious RNA isolation

Robust performance—Results equivalent to purified RNA

Broad input dynamic range—Linear detection from 10 to 100,000 cells

• 1000 HeLa cells. RT and subsequent PCR reactions were done for 144 miRNAs. 111 had a CT below 35 and plotted on the graph.

y = 0.9765x + 1.4269R2 = 0.9307

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24

26

28

30

32

34

36

38

22 24 26 28 30 32 34 36 38Purified RNA CTs

Cel

ls-to

-CT

lysa

te (C

T)


NEW - TaqMan® miRNA ABC Kit

Targeted microRNA Isolation for Expression Analysis– Achieved by hybridization of miRNAs to anti‐miRNA oligos attached to

Dynabeads®

– Anti-miRNA oligos correspond to the assays in the TaqMan® Array Human MicroRNA A and B Cards

Entire workflow is typically 75 minutesBeads have capacity to capture 128-380 fmol of each miRNA


TaqMan® miRNA ABC Kit: Data using Human Panel A Kit

Purified MicroRNA from serum, plasma and FFPE samples were analyzed using a set of TaqMan® microRNA (single tube) Assays− Each bar represents duplicate

sample preparations and quadruple PCR replicates.

Excellent Reproducibility


Pri-miRNA, miRNA & All Non-coding RNA TaqMan® Assays

TaqMan® Assays provide a highly reliable way to measure expression of various RNA transcripts.

Provides high sensitivity, specificity, dynamic range & superior data quality

Delivers faster time-to-results

Requires no design expertise

Works under universal cycling conditions

Detects only Pri-miRNA, miRNA or non-coding RNA transcripts

TaqMan® MicroRNA

Assays

TaqMan® Pri-miRNA

Assays


MicroRNA Discovery with RNA-Seq

RNA sequencing is the leading hypothesis-free method for microRNA analysis & enables the discovery of new miRNAsThe number of novel miRNAs discovered has increased exponentially since the onset of Next Gen Sequencing

0

5000

10000

15000

1.1

1.3

1.5

2.1 3 4

5.1 7 8

8.2

9.1

10.1 12 14 16 18

Num

ber of Entries

miRBase Version

Sanger miRBase Growth

Distribution Run time Throughput

Ion Torrent(PGM™)

Wide – low barrier to entry, affordable system

2 hours – 35 bases & barcodes

3 barcoded small RNA samples (Ion 318™ Chip)


RNA Preparation from Stabilized Blood


Blood-to-Ct™ kit pre-screening and MagMax™ purification

Take 500µL aliquot

Tempus® Stabilized Blood‐to‐CT™ kit*

Perform selected qRT-PCR assays on lysatesto select only tubes of interest:

Do full RNA purification only from these tubes.

Eliminate irrelevant samples

Tempus® MagMAX™ for Stabilized Blood Tubes Kit*

Process 12-96 samples in <1 hour

* Compatible with Tempus® Blood RNA for research use only


Reverse Transcription

Real-Time PCR

60 minute mark for 12 tubes 2 hr 12 min

Total for 12 tubes

Existing RNA Extraction Protocol


Collected blood

Aliquot 500 ul

Pellet and Wash 2X

Incubate 8 min at room temp.

Add Stop Solution

Reverse Transcription

Real-Time PCR

2 min. at room temp.

Stabilized Blood-to-CT™ Protocol (tubes and plates)

60 minutes for 12 or 96 samples in comparison to 2 (12) or 4 hrs (96)

Resuspend in Digestion Solution


5

10

15

20

25

30

35

40

5 10 15 20 25 30 35 40Pure Avg CT

Lysa

te A

vg C

1.5 mL Tube

96-well PlateR2 = 0.97

R2 = 0.97R2 = 0.95

mRNA miRNA

Comparable results to pure RNA


Conclusions

Stabilized blood− Provides immediate stabilization of host gene transcripts− Allows for delay between collection and analysis− Allows for long term storage of samplesStabilized Blood-to-CT™ Nucleic Acid Preparation Kits for qPCR

Enables expression analysis directly from a 500 μL aliquot of stabilized human whole blood without conventional RNA purification.

MagMAX™ for Stabilized Blood RNA Islolation KitsPurify high quality total RNA, including microRNAs, from blood RNA tubes.


TaqMan® Single Cell-to-Ct™ kits


• 4 functional steps: cell lysis, reverse transcription, cDNA pre-amplification, and Real-Time PCR

• gDNA removal occur at the same time as cell lysis - 5 minutes at room temperature

• All done in same tube (no loss due to tube transfers)

• Enable use of entire lysate sample in subsequent RT/PreAmp rxns

Single Cell-to-Ct™ kit: a complete workflow to obtain statistically relevant single cell data

• Performance benefit with smaller volumes and allows use of 384-well plates

• Increased sensitivity (SuperScript III)• Optimized volumes in workflow

minimize errors in reaction assembly (no water addition in any step)

• Multiple stopping points• microRNA and DNA protocols


Product Performance

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16

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20

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24

26

28C

T

1 Cell Equivalents

Single Cells 100 cells

20.4

21.6

13.6

N=84 single cells


Conclusions and Impacts

• There are significant differences from cell-to-cell

• Single cells have a large range of gene expression levels.

• Analyzing gene expression en masse gives an average profile and masks differences and variability (gene-to-gene and cell-to-cell)

• Small populations are lost when large populations are averaged

• Average expression patterns of single cell populations are similar to 100 cells.


The TaqMan® Single Cell-to-Ct™ kit

• Optimized reagents provide a simplified workflow for expression analysis of single cells by qRT-PCR

• Enables transfer of entire cell into each step

• No sample is lost during the reaction which occurs in a single tube

• Sensitivity is maximized by use of reagents such as SuperScript III and PreAmp Master Mix

• Variation introduced by the protocol is very low

• Enables the acquisition of statistically significant data sets


FFPE Samples

Formaldehyde fixation maintains tissue structure

Most common tissue storage method used today by pathologists

Central to cancer and disease research, biomarker discovery and personalized medicine

Used currently in conjunction with qRT‐PCR or qPCR studies but it is branching out to include:

− Microarrays

− Methylation studies

− miRNA work

− Sequencing


Challenges with FFPE Samples

Low Yield

‐Nucleic acid is both trapped and modified by extensive protein‐protein and protein‐nucleic acid crosslinks.

Chemical Modification and Fragmentation

‐High temperatures required for paraffin infiltration can accelerate chemical reactions which modify nucleic acid

‐Fragmentation and progressive degradation of nucleic occurs during long‐term storage due to chemical modifications

‐Nucleic acid is often chemically modified to a degree that renders it incompatible with many molecular analysis techniques

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Noticeable differences between FFPE and Unfixed

Unfixed FFPE

Agilent Nanochip Results


MagMAX™ FFPE Isolation Kits ‐ Summary

• A high‐throughput total nucleic acid purification solution from FFPE samples.

• Isolation of both Total RNA and DNA

• Magnetic bead‐based isolation in 96‐well plate format to increase throughput

• Simplified automated workflow completed in 30 minutes.

• Less‐toxic workflow by eliminating xylene


Between method/tissue concordance ‐ PGM

fresh frozen tumor log2(RP100K)

RP100K = counts per 100k mapped reads (miRBase 16)

fresh frozen

NATlog2(RP1

00K) spearman= 0.843

FFPE tumor log2(RP100K)

FFPE

NATlog2(RP1

00K)

spearman= 0.840

spearman= 0.873

FFPE

tumor

log2(RP1

00K)

fresh frozen tumor log2(RP100K)

spearman= 0.860

fresh frozen NAT log2(RP100K)

FFPE

NAT log2(RP1

00K)

Observation: source of most variation mostly (and subtly) from tissue type and not the tissue preparation/storage method

Present miRNAs only (row sum across all runs >=10 counts)


• tiny microvesicles with nucleic acid and protein “cargo”

• secreted by all cells

• found in most body fluids• Blood, saliva, urine, breast milk and

cell culture media

• functions are diverse• Cellular communication

• Clean up cell’s trash

What are Exosomes and what do they do?


Ultracentrifugation is the current “Gold Standard” isolation method for exosomes

Proven methodProduces clean preps

But…Requires a large amount of starting materialYield can be very lowHighly labor intensive and time consuming − 2-3 days depending on sample type,

sample amount and if using gradient or cushion


Isolation of exosomes

Purification of exosomal RNA &

protein

Analysis of exosomal RNA &

protein

Exosome Immunoprecipitation (Protein A or Protein G)

Optimized Exosome workflow


Protein Analysis from Exosomes:Further purification using Immuno-magnetic isolation


Human serum-derived exosomal RNAisolated using Total Exosome Isolation Reagent

• Differential RNA representation of specific miRNAs

• Quantities normalized by the percentage of reads aligned to that transcript out of the total mapped.

• Mapped read count distribution by RNA species

Cell Free Plasma Exosome Isolation Reagent

Donor 1 Donor 2 Donor 1 Donor 2

Cell Free Plasma Exosome Isolation Reagent


The Ion RNA‐Seq Offering

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• Preserves strand information

• Small RNA and/or WT libraries

•Optimized workflow

• Template preparation in just 3h with only minutes of hands‐on time

• 200bp read‐lengths

• Intuitive microarray‐like UI and workflows developed for Ion Torrent

RNA purification Library construction Sequencing Data analysis


RNA-Seq WorkflowSamples to results in a single day

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2.5HOURS

mRNA

1HOUR

miRNA


• Library construction in ~ 6 hours• Sequence in as little as 1 hour for small RNAs and 2.5 hours for mRNAs

• 3 Ion Chips to tailor the number of reads by just changing the chip

• 16 barcodes for sample multiplexing• 35 to 200 base read length for sequencing miRNA or mRNA

• Easy library construction with automation friendly magnetic bead clean up steps to replace columns

• No gel size selection needed for small RNA libraries

Simple, Scalable, Fast Ion RNA-Seq v2mRNA and miRNA

Simplicity

Scalability

Speed

46

http://products.invitrogen.com/ivgn/product/4462923


Ion Total RNA-Seq Kit v2: Simple & Fast Workflow

Confidential and Proprietary—DO NOT DUPLICATE47

Preparation of small RNAObtain total RNA then determine the quality

Enrich small RNA

Quantitate small RNA sample and determine input amount

Preparation of whole transcriptome RNA25-500 ng rRNA-depleted total RNA or 1-500 ng poly(A)

RNA or 100 ng total RNA

Fragment the RNA

Clean up the RNA

SOLiD™ amplified library constructionHybridize and ligate the RNA adapters

Perform reverse transcription

Purify the cDNA

Assess the yield and size distribution of the amplified DNA

SOLiD™ System templated bead preparation and sequencing

Preparation of small RNAPreparation of whole transcriptome RNA

PGM ™ amplified library construction

Amplify the cDNA (BC addition)

Purify the amplified DNA

IonSphere™ particle preparation and Ion PGM™sequencing

Magnetic beads

Magnetic beads

Magnetic beads

Magnetic beads

Magnetic beads

Magnetic beads

< 6 hours< 5 hours


Strong Correlation of Ion Proton™ Data with qPCR MAQC Data

TaqM

an™

R=0.958N=657 assays

(UHR/HBR)


Detection of Novel Exons & Alternate Splicingwith Ion PGM™ Sequencer data

Confidential and Proprietary—DO NOT DUPLICATE49

Ion PGM™ sequencer derived RNA-Seq results from analysis of a Ewings sarcoma cell line (data courtesy T. Triche, Childrens Hospital of Los Angeles).


Comprehensive quantitation of PGM reads using an iterative mapping strategy

White paper is available on the Ion Community that explains more details on processing Ion RNA-seq data


Single kit for preparation of either small RNA or whole transcriptome libraries

Maintains strand orientation

Start with as little as 1 ng poly(A) RNA or 25 ng of rRNA-depleted RNA isolated from 100 ng total RNA, or 1 ng enriched miRNA

Easy, fast (6 hr) library construction with automation friendly magnetic bead clean up steps

No gel size selection needed for small RNA libraries

Compatible with Ion Xpress™ RNA-Seq Barcode Kit 1-16

Benefits of Ion Total RNA-Seq Kit v2

Adaptor ligation

Reverse transcription

Amplification

Library Construction

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For Research Use Only. Not for use in diagnosticprocedures.

© 2012 Life Technologies Corporation. All rights reserved.

The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan is a

registered trademark of Roche Molecular Systems, Inc.

CyBi is a registered trademark of CyBio AG. Zephyr is a registered trademark of Caliper Life Sciences, Inc. Biomek is a registered

trademark of Beckman Coulter, Inc. Freedom EVO is a registered trademark of Tecan Group Ltd.


RiboMinus™ Eukaryote System v2Removing cytoplamic and mitochondrial rRNA

Low Input RiboMinus™ Eukaryote System v2

Includes Mag Bead Cleanup

Or

RiboMinus™ Eukaryote System v2

Includes Mag Bead Cleanup

or

RiboMinus™ Eukaryote Kit v2Cleanup Columns, available separately


New RiboMinus Eukaryote for RNA-Seq V2

5S

5.8S

18S 28S

HeLa (RIN 9.8) Human Kidney (RIN 6.9)

18S

28S

More efficient removal of ribosomal RNA (rRNA). Effective with compromised RNA. Demonstrated effective prior to RNA‐Seq library generation!


PGM™ 318 Chip Sequencing Count Distribution

HeLa rRNA depleted Libraries

Mapping Statistics for UHRR rRNA depleted Libraries

rna sample preparation and analysis tools from ambion...

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