Northern Blotting

By: Dr. Ashish Patel,

Ph.D.Scholar, Animal Genetics & Breeding

• The northern blot is a technique used to study gene expression by detection of RNA in a sample. Or

• A northern blot is a method used to detect specific RNA molecules among a mixture of RNA and also be used to analyze a sample to measure the RNA expression of particular genes.

• The name of northern blot method derived from its similar first blotting technique known as a Southern blot (which was invented by Edwin Southern).

• Northern blotting was developed by Alwine in 1977.

Principles of Northern blotting

• Electrophoresis – Seperates RNA on the basis

of their Molecular weight and type in agarose

gel which have EtBr ,an intercalating agent in it.

• Capillary action – RNA bands move towards

blotting paper by capillary movement and

entrap in sheet and buffer moves ahead.

Steps involving Northern Blotting• RNA isolation• Separation of RNA using Gel Electrophoresis • Blotting• Hybridization and Washing of excess probes • Visualization• The Northern Blot procedure is quite similar to

that of Southern blot, except that the biomaterial used is RNA instead of DNA.

1. RNA ISOLATION:• The RNA is isolated from the cell.• All protocols, techniques, and commercially

available kits used to isolate RNA with common attributes:

Cellular lysis and membrane disruptionInhibition of ribonuclease activityDeproteinizationRecovery of intact RNA

Methods of RNA extraction1. RNA isolation by acid guanidinium

thiocyanate-phenol-chloroform extractionPrinciple:

RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation.

Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain in the lower organic phase.

Total RNA is then recovered by precipitation with isopropanol and can be used for several applications.

2. RNA isolation by Column-based TechnologyPrinciple:

Nucleic acid will bind to the solid phase of silica under certain conditions.

A buffer solution along with sample, ethanol or isopropanol forms the binding solution and the binding solution is transferred to a spin column and the column is put in a centrifuge. The centrifuge forces the binding solution through a silica gel membrane that is inside the spin column and nucleic acid will bind to the silica gel membrane

3. RNA isolation by Chemicall cum Column-based Technology

Quality assessment of total RNA

• RNA quality can be assess by Nano Drop technology or by Bioanalyser

• There are three quality controls that are performed on isolated RNA.

Via Nanodrop:• Quantity of RNA: measure in ng/µl.• Purity of RNA: The ratio of the absorbance at 260 and

280 nm is used to assess the RNA purity of an RNA preparation. Pure RNA has an A260/A280 of ranges 1.8 to 2.0.

• Integrity of the RNA: can be measured on bioanalyzer

• Bioanalyzer: perform capillary electrophoresis and uses a fluorescent dye that binds to RNA to determine both RNA concentration and integrity.

• it is recommended to use at least 50 ng/μl for a meaningful RNA Integrity Number (RIN).

• The RNA Integrity Number (RIN) software algorithm allows the classification of total RNA, based on a numbering system from 1 to 10, with 1 being the most degraded and 10 being the most intact.

• If RIN of 5 might not work for a microarray experiment, but might work for an RT-PCR experiment.

• In general, RINs higher that 7-8 seem to be working well in most of experiments.

• RINs below 7 require extra validation studies before we are able to conclude “how bad is still good enough".

2. SEPARATION OF RNA• Once RNA samples are isolated, the next step is denaturing

agarose gel electrophoresis. • Types of gel: Agarose (for DNA and RNA: source: sea weed),

Polyacrylamide gel (for Protein) and Starch (for Protein).• Gel Conditions: • 1. Denaturing gels: Disrupt the natural structure of DNA /

RNA and causing it to unfold into a linear chain. Thus, the mobility of each macromolecule depends only on its linear length and its mass-to-charge ratio.

• Thus, the secondary, tertiary, and quaternary levels of biomolecular structure are disrupted, leaving only the primary structure to be analyzed.

• Nucleic acids denatured by urea/formaldehyde in the buffer, while proteins are denatured using sodium dodecyl sulfate.

• 2. Native gels: non-denaturing conditions, so that the analyte's natural structure is maintained.

• In Northern blotting, Formaldehyde has been the denaturant traditionally used during electrophoresis.

• Formaldehyde agarose gel electrophoresis is normally used in the separation of RNA as formaldehyde agarose gel prevent RNA from folding on itself.

• The disadvantage of using formaldehyde is the need to pour and run gels in a fume hood.

• On electrophoresis RNA molecules moves towards positive pole as RNA is negatively charged.

Choice of buffer for gel electrophoresis

• Factors for choosing a buffer:• A simple buffered solution contains a mixture of a weak acid (HA) and

its conjugate (A-) base.1. Formal charges of buffer species: - Generally, buffers which form

ions of high charge magnitude (+2, +3, -3, etc.). Resulting in to at relatively low concentrations, the gel conducts too much current. Furthermore, with ions moving quickly through the gel, the buffer may become depleted. One of the reasons Tris-borate is a popular buffer for electrophoresis is that both Tris base and borate are uncharged part of the time at the desired pH, which reduces their electrophoretic mobility.

2. Molecular size - Low charge Tris base moves slowly in electrophoresis because of its relatively large molecular size.Having a low charge to mass ratio, Tris moves much more slowly than small ions such as chloride or phosphate.

3. pKa value (Acid dissociation constant) - A buffer should be chosen with a pKa that is very close to the desired pH.Other factors to consider when choosing a buffer would include toxicity, solubility, UV absorption and the possibility of interaction with other species present in the solution.

There are a number of different buffer configurations that are used for different kinds of electrophoresis

Homogenous Buffer System - the identity and concentration of buffer components are the same in the gel and both tanks. This is used for most forms of DNA and RNA electrophoresis.

Buffer Molecular Weight (Mr) pKa

Acetic Acid 60.05 4.8Boric Acid 61.68 9.23Glycine 75.07 9.8Tricine 179.18 8.15Tris 121.1 8.06

• Multiphasic Buffer Systems - System uses differing buffers and is used for SDS-PAGE (often called the Laemmli system). The Laemmli system uses an additional gel layer above the separating gel.

Buffers used are:Stacking Gel - Tris-HCl, pH 6.8, Separating Gel - Tris-HCl, pH 8.8 at a higher concentration, Tanks - Tris-glycine, pH 8.8.

• Buffer Additives - are usually added to the buffer perfusing the gel and can include:Hydrogen bonding agents: Urea and formamide, which disrupt hydrogen bonds, that affect the conformation and solubility of molecules.Surfactants : Triton X-100, Tween 20 or SDS. SDS is the most commonly used detergent. This causes the protein chains to unwind from their native configuration and the protein is said to be denatured.

Reducing Agents : 2-mercaptoethanol or dithiothreitol that break the disulphide bond linkages that hold protein chains together. The protein is then said to be reduced.

3. BLOTTING• Simply blotting is process of transferring the RNA molecules to the

nitrocellulose membrane or nylon membrane:Choice of membrane:• There are several types of commercially available membranes suitable

for RNA analysis, composed of different materials and carrying different charges.

• The common ones are made of nylon and nitrocellulose, and may be neutral, negatively or positively charged.

• Nylon (polyamide) membranes are made of the most durable material, but can shrink or warp if exposed to organic solvents.

• Nitrocellulose tends to tear easily in washing steps and becomes very fragile.

• Negative membranes give the cleanest background, but result in poor specific signal.

• Positively charged membranes give the best signal of all, but they also result in higher background.

• Many scientists feel nylon is better since it binds more and is less fragile.

• Three types of blotting membranes are available:Membrane

Properties Applications Pore sizeReprobing

Nitrocellulose

Most widely used membrane for western blottingGood binding capacityProteins bind to the membrane due to hydrophobic interactionsProtein binding capacity: 80 µg/cm2

Western transferAmino acid analysisSolid phase assay systems

0.2 µm0.45 µm

No

PVDF Higher binding capacity than nitrocelluloseStrong hydrophobic character and solvent resistantPhysically stronger than nitrocelluloseCompatible with commonly used protein stains and immunodetection methodsProtein binding capacity: 50-150 µg/cm2

Protein sequencingWestern transferAmino acid analysisSolid phase assay systems

Yes

Nylon Microporous membrane modified with strongly basic charged groupsIdeal for binding negatively charged biomolecules such as DNA and RNALow background for enhanced resolutionMembrane is formed around a non-woven polyester fiber matrix which confers high tensile strength, toughness, and flexibility

Southern and northern transfersSolid phase immobilizationDry chemistry test stripsEnzyme immobilizationGene probe assays

0.45 µm Yes

• Once separated by denaturing agarose gel electrophoresis, the RNA is transferred to a positively charged nylon membrane and immobilized for subsequent hybridization.

• For fast, reproducible transfer, the iBlot Dry Blotting System offers complete transfer of RNA to nylon membrane typically in 7 minutes. With dry blotting, there is no need for additional buffer or liquids that can introduce variability into the result.

• This system is compatible with both polyvinylidene difluoride (PVDF) and nitrocellulose membranes, and has comparable performance to traditional wet transfer methods in a fraction of the time.

• High detection sensitivity• Increased blotting reliability and reproducibility• Flexible gel-size formats and membrane types• High-quality and more compact transfer stacks

iBlot Dry Blotting System

Semi-drytransfer

Wet or semi-wet transfer

Buffer preparation 0 minutes 30 minutes 30 minutes

Soaking gel in transfer buffer 0 minutes 20 minutes 0 minutes

Assembling layers 2 minutes 10 minutes 10 minutes

Transfer 7 minutes 45–90 minutes 1–3 hr

Cleanup 0 minutes 10 minutes 10 minutes

Total elapsed time 9 minutes 1 hr, 55 min–2 hr, 40 min

1 hr, 50 min–3 hr, 50 min

Time saved with the iBlot Dry Blotting System

— 1 hr, 45 min– 2 hr, 30 min

1 hr, 40 min– 3 hr, 40 min

Comparison of elapsed time for protein transfer with the iBlot Dry Blotting System to other blotting methods.

iBlot Dry Blotting System Semi-dry transfer Wet or semi-wet transfer

Preassembled stacks ready for protein transfer containing electrodes, buffer matrices, and PVDF or NC membrane

Transfer stack (both top and bottom) composed of sponge and filter paper, soaked in buffer

Transfer stack composed of sponge and filter paper, soaked in a tank filled with buffer

iBlot Dry Blotting System

Semi-dry transfer Wet or semi-wet transfer

Transfer buffer required?

No 100–250 mL, or just enough to construct a bubble-free sandwich

1–1.5 L, or enough to fill the transfer tank

Transfer time 7 min, plus 2 min transfer preparation

45–90 min, plus 70 min preparation and assembly

1 hr–overnight, plus 50 min preparation and assembly

Transfer quality •Reproducible and good transfer quality for proteins between 11 and 220 kDa.

Variable and inefficient transfer quality:Reduced buffer capacity limits transfer time, especially for mid- to large molecular weight proteinsMembrane and filter paper MUST be cut to exact gel size, otherwise current will short-circuit around the edge of the gel

•Reliable and good transfer quality: Increase temperature during blotting, unless buffer is mixed and cooled during blotting•High-current power source is typically required for 1–2 hour transfer

Supple. equipment required

None External power supply External power supply

Post-transfer requirement

None •Wet-soaking filter paper for clean-up•Salt deposits on electrodes require regular maintenance

•Large amount of hazardous buffer to discard•Wet-soaking sponges for clean-up•Salt deposits on electrodes require regular maintenance

4. STABILIZATION• Once the RNA has been transferred to the membrane, it is immobilized

through covalent linkage to the membrane by two methods.1. UV crosslinking is one of the most popular methods, using either a hand-held

UV lamp at short wavelength, or a commercial crosslinking device. • Shortwave UV light causes the uracil base to become highly reactive and to

form covalent linkages to amine groups on the surface of the membrane. • The "auto-crosslink" feature on commercially available, calibrated UV

crosslinkers.• If a calibrated instrument is not available, it is possible to use standard

laboratory equipment such as transilluminators and handheld ultraviolet lamps to fix RNA targets to a membrane.

• Care must be taken not to under or overexpose the RNA to UV light — both of which will decrease hybridization signals. Usually a one minute exposure with 254 nm light.

• The other common method baking the membrane in an oven at 80°C

2. Baking works by heating the membrane to drive out all water solubilizing the RNA.

• A large component of RNA is its hydrophobic nucleotide bases, which make hydrophobic contacts with aromatic groups on the membrane.

• This interaction is affected by heating in an oven at 80°C for 15 min.

• The only danger in baking is that the membrane can be damaged if the heat is not regulated to prevent temperatures from rising much higher than 100°C.

Probes

• Probes for northern blotting are composed of nucleic acids with a complementary sequence to all or part of the RNA of interest, they can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence.

• RNA probes should be withstand during more rigorous washing steps preventing some of the background noise.

• Commonly cDNA is created with labelleled primers for the RNA sequence of interest to act as the probe in the northern blot.

• The probes must be labelled either with radioactive isotopes (32P, 33P, or 35S. Radioactive labeling provides the most sensitive method for detection, allowing detection of 0.01 pg.) or with chemiluminescence in which alkaline phosphatase or horseradish peroxidase (HRP) break down chemiluminescent substrates producing a detectable emission of light.

• The chemiluminescent labelling can occur in two ways: either the probe is attached to the enzyme, or the probe is labelled with a ligand (e.g. biotin) for which the ligand (e.g., avidin or streptavidin) is attached to the enzyme (e.g. HRP).

• X-ray film can detect both the radioactive and chemiluminescent signals and chemiluminescent signals are faster, more sensitive, and reduce the health hazards.

Detection radioactive labelled probe (Disadvantages of 32P): • Short half life (about 2 weeks) means probes must be used immediately, and the

labeling reagent cannot be stored for long. • Contamination problems: all materials and equipment must be dedicated to

radioactive work only. Regular lab-wide testing for contamination is required. • Expense of disposal of radioactive waste. • Must have access to a dark room to set up and develop films. Nonradioactive detection system are colorimetric, fluorescent and chemiluminescentColorimetric detection generally involves the production of a colored precipitate

which can be seen with the naked eye. In a typical system, the DNA probe itself is labeled with an antigen such as Digoxigenin and after hybridization to its target it would be exposed to an anti-digoxigenin antibody conjugated to an enzyme capable of catalyzing a colorimetric reaction.

Fluorescent detection involves probes which are directly labeled with fluorophores, or indirectly. For example, if probe is labeled with biotin, it would be exposed to avidin or streptavidin fluorescent tag and Fluorophores emit light at an appropriate wavelength.

Chemiluminescence is a combination of these two: an enzymatic reaction that triggers the release of ordinary visible light.

5. HYBRIDIZATON AND WASHING OF EXCESS PROBS• Hybridization with radiolabelled or fluorescently labelled probe.Prehybridize before hybridization:• Blocks non-specific sites to prevent the single-stranded probe from

binding just anywhere on the membrane.Hybridization:• Incubate membrane with labeled RNA probe with target sequence:

Probe could be lablleled with 32P, biotin/streptavidin probe. • Probes for northern blotting are composed of nucleic acids with a

complementary sequence to all or part of the RNA of interest, they can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence.

• The probes must be labelled either with radioactive isotopes (32P) or with chemiluminescence.

• Hybridization between probes and the target RNA• Washing of excess probes

6. VISUALIZATION

Autoradiography:• Place membrane over X-ray film.• X-ray film darkens where the fragments are complementary

to the radioactive probes.

NORTHEN BLOT APPLICATION

• Northern blots are particularly useful for determining the specific genes are being expressed at mRNA level.

• Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, pathogen infection, and over the course of treatment

• The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue, as well as the gene expression in the rejection of transplanted organ.

Comparison of Northern, Southern and Western blotting

Thank You