Hapuarachchige Dona Thisarani Sankalpana Samarasinghe

Diploma in Bio Medical Science

June 2016

Northern Blotting

› The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA

› The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.

› The Northern blot, also known as the RNA blot, is one of the blotting techniques used to transfer DNA and RNA onto a carrier for sorting and identification.

› The Northern blot is similar to the Southern blot except that RNA instead of DNA is the subject of analysis in this technique.

What is Northern Blotting?

› It is mRNA which is isolated and hybridized in northern blots› The formaldehyde was use in electrophoresis gel as a

denaturant because the sodium hydroxide treatment used in the Southern blot procedure would degrade the RNA.

› The Northern blot is useful for the study of gene expression in two ways.

› First, the position of bands on the blot provides a direct measure of RNA size.

› Knowing the size of the RNA will provide an estimate for the transcript’s coding capacity and thus the size of the protein it encodes.

Applications:

› Second, the Northern blot analysis of RNA samples from many different tissues enables researchers to determine which specific tissue a gene is expressed in along with the relative levels of its expression in all cells where transcription is occurring.

› The Northern blot is a valuable method used by researchers in determining gene expression patterns.

› For example, many scientists researching Huntington disease or breast cancer are able to determine the expression patterns of the genes responsible for these diseases using blotting techniques.

1.RNA isolation:• This part of the Northern Blot is an important step because high

quality,mRNA is extracted from the cells and purified.

2. Probe generation:• The mRNA is loaded onto a gel for electrophoresis. • Lane 1 has size standards (a mix of known RNA fragments) Lane 2

has the RNA.• Northern blots can be probed with radioactively or nonisotopically

labeled RNA, DNA or oligodeoxynucleotide probes.

Process:

3.Denaturing agarose gel electrophoresis: • Formaldehyde has traditionally been used as the denaturant,

although the glyoxal system has several advantages over formaldehyde.• An electric current is passed through the gel and the RNA moves

away from the negative electrode. • The distance moved depends on the size of the RNA fragment.• Since genes are different sizes the size of the mRNAs varies also.• This results in a smear on a gel.• Standards are used to quantitate the size. • The RNA can be visualized by staining first with a fluorescent dye

and then lighting with UV.

4. Transfer to solid support and immobilization: • RNA is single-stranded, so it can be transferred out of the gel and onto a

membrane without any further treatment.• The transfer can be done electrically or by capillary action with a high salt

solution.

5. Prehybridization and hybridization with probe: • A labelled probe specific for the RNA fragment in question is incubated with

the blot. • The blot is washed to remove non-specifically bount probe and then a

development step allows visualization of the probe that is bound.

6. Washing: • The probe is bound specifically to the target mRNA and that

there is negligible non-specific binding to other mRNA or the nylon membrane itself.

7.Detection:• If a radiolabeled probe was used, the blot can be wrapped in

plastic wrap to keep it from drying out and then immediately exposed to film for autoradiography. • If a nonisotopic probe was used, the blot must be treated with

nonisotopic detection reagents prior to film exposure.› Hybridization signals are then detected.

Northern blotting

› Remove air bubbles trapped between the gel & the membrane.› Ensure that all buffer components are fully dissolved before

using.› Check whether the membrane has damaged at cut adges.› Ensure that the electrophoresis tanks are rinsed with distilled

water after used.› Control the temperature during hybridization› Always check for the incorporation of the radioactive label

before using the probe.› Don’t reuse electrophoresis buffer & radioactive probes.

Precautions for blotting

1) Increase the Amount of RNA Loaded in Each Lane.2) Use Poly(A) RNA Instead of Total RNA.• 10 µg of mRNA is equivalent to 300–350 µg of total RNA.

(mRNA comprises only about 0.5–3% of total RNA.3) Switch From a Traditional Hybridization Buffer to ULTRAhyb.• Ultrasensitive Hybridization Buffer can increase the sensitivity

of a blot hybridization up to 100-fold. 4) Use High Specific Activity Probes. The specific activity of the

probe should be at least 108 cpm/µg

8 Ways to Increase the Sensitivity of Northern Hybridizations

5) Use an Optimal Hybridization Temperature. • Temperatures of 42°C for DNA probes and 68°C for RNA

probes usually give excellent results in 50% formamide-based hybridization buffers.

6) Use Freshly Synthesized Radiolabeled Probes. • High specific activity probes degrade rapidly due to

autoradiolysis, resulting in low signal and/or high background.7) Increase Exposure Time8) Follow the Manufacturer’s Recommendations to Crosslink

the RNA to the Membrane.

› Analysis of gene expression can be done by several different methods including RT-PCR, microarrays, serial analysis of gene expression (SAGE), as well as northern blotting.

Advantages and Disadvantages

› Microarrays are quite commonly used and are usually consistent with data obtained from northern blots; however, at times northern blotting is able to detect small changes in gene expression that microarrays cannot.

› The advantage that microarrays have over northern blots is that thousands of genes can be visualized at a time, while northern blotting is usually looking at one or a small number of genes.

› The chemicals used in most northern blots can be a risk to the researcher, since formaldehyde, radioactive material, ethidium bromide and UV light are all harmful under certain exposures.

› Compared to RT-PCR, northern blotting has a low sensitivity, but it also has a high specificity, which is important to reduce false positive results.

› The advantages of using northern blotting include the detection of RNA size, the observation of alternate splice products, the use of probes with partial homology, the quality and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and reprobed for years after blotting.

› Researchers occasionally use a variant of the procedure known as the reverse northern blot.

› In this procedure, the substrate nucleic acid is a collection of isolated DNA fragments, and the probe is RNA extracted from a tissue and radioactively labelled.

› Thus the reverse procedure, though originally uncommon, enabled northern analysis to evolve into gene expression profiling, in which many of the genes in an organism may have their expression monitored.

Reverse northern blot

Thank you