History of Northern blotting

NORTHERN BLOTTING History of Northern blotting[1] • • •

Is one type of blotting for the RNA detection with labeled DNA probes. It was developed in 1977 by Alwine et al. Stanford University. It was named after the Southern blot technique which blots for DNA and was invented by Edwin M. Southern in 1975.

Background information of Northern Blotting Technique[1] •

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Northern analysis despite its age in the high tech world of polymerase chain reaction (PCR), nuclease protection assays (RPAs) and microarrays, is still the gold-standard for the detection and quantitation of mRNA levels. The reason is it allows a direct comparison of the messenger RNA (mRNA) abundance (amount of mRNA present in sample) between samples on a single membrane. The main difference of Northern blotting from other techniques is that RNA is the factor that used to be detected. RNA is separated out by:  RNA gel electrophoresis (usually argarose)  Subsequent transfer to membrane  Hybridization with probe  Detection For the hybridization probes it may be DNA or RNA in northern blotting similar to Southern Blotting. A variant of the procedure known as the reverse northern blot was occasionally (although infrequently) used. In this procedure, the substrate nucleic acid (that is affixed to the membrane) is a collection of isolated DNA fragments, and the probe is RNA extracted from a tissue and radioactively labelled. The used of DNA microarray have come into widespread use in late 1990 and early 2000 is akin to the reverse procedure. They involve the use the use of isolated DNA fragment affixed to a substrate, and hybridization with a probe made from cellular RNA. Thus the reverse procedure, though originally uncommon, enabled the one-at-a-time study of gene expression using northern analysis to evolve into gene expression profiling, in which many (possibly all) of the genes in an organism

Diagram of western blot procedure [1]

Northern blot principle[1] 1. RNA isolation 2. Gel electrophoresis of RNA for separation 3. Transfer to membrane (usually positively charged nylon as RNA is negaticely charged) 4. Cross-linking of RNA to membrane (usually by UV-crosslinking or chemical means) 5. Hybridization 6. Detection

Procedure [2] 1. RNA is isolated from several

biological samples (e.g. various tissues, various developmental stages of same tissue etc.) * RNA is more susceptible to degradation than DNA.

2. Sample’s are loaded on

gel and the RNA samples are separated according to their size on an agarose gel .  The resulting gel following after the electrophoresis run.

1. The gel is then blotted on a nylon membrane or a nitrocellulose filter paper by creating the sandwich arrangement.

Diagram of blotting sandwich [4]

2. The membrane is placed in a dish containing hybridization buffer with a labeled probe. •

Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest

The probe can be either ss-DNA or RNA

1. The membrane is washed to

remove unbound probe.

Access probe is removed 2. The labeled probe is detected

via autoradiography or via a chemiluminescence reaction (if a chemically labeled probe is used). In both cases this results in the formation of a dark band on an X-ray film. • Now the expression patterns of the sequence of interest in the different samples can be compared. *Improvement in sensitivity of this method can be achieved by using high specific activity antisense RNA probes (step 4), optimized hybridization buffer (step 4), and positively charged nylon membrane (step 3) [2] *RNA ISOLATION This part of the Northern Blot is an important step because of high quality, intact RNA needs to be obtained. There are several ways of the isolation can be performed. However, common attributes include cellular lysis and membrane disruption, inhibition of ribonuclease activity, deproteinization and recovery of the intact RNA. [3]

Applications of the northern blot[1] Have been superseded in most areas by Real Time PCR and microarray approaches but it is not often used in clinical and diagnostic purposes. The northern blot principle and its variations are used however in molecular biology research to: • • • • •

A gold-standard for the direct study of gene expression at the level of mRNA (messenger RNA transcript) Detection of mRNA transcript size Study of RNA degradation Study of RNA splicing (can detect alternatively spliced transcript) Study RNA half life

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Study IRES (internal ribosomal entry site) –to remove possibility or RNA digestion vs second cistron translation Often used to confirm and check transgenic / knockout mice (animals) *transgenic – containing genetic material into which DNA from a different organism has been artificially introduced.

Advantages of northern blotting[1] • • • • • •

Widely accepted and well regarded method It is a straight-forward method Often it is used as a confirmation or check Often a gold-standard It is a versatile principle which allow the usage of many types of probes (vs Real Time PCR) including : radiolabeled and non-radiolabeled, in vitro transcribed RNA and even oligonucleotides such as primers. Sequences with even partial homology, unlike real time PCR or methods can be used as hybridization probes (sequence from different species for homology analysis, or even genomic fragments can be used)

Disadvantages of northern blotting technique[1] • •

The whole of northern blotting process takes a long time, from the preparation through to detection. The standard of northern blot is less sensitive compared to the nuclease protection assays and Real Time PCR. The sensitivity may be increased with the use of nylon positively-charged, use of a highly specific antisense probe.