RNA Blotting

The same basic process of nucleic acid blotting can be used to transfer RNA from gels on to similar membranes. This allows the identification of specific mRNA sequences of a defined length by hybridisation to a labelled gene probe and is known as Northern blotting. With this technique it is not only possible to detect specific mRNA molecules but it may also be used to quantify the relative amounts of the specific mRNA. It is usual to separate the mRNA transcripts by gel electrophoresis under denaturing conditions since this improves resolution and allows a more accurate estimation of the sizes of the transcripts. The format of the blotting may be altered from transfer from a gel to direct application to slots on a specific blotting apparatus containing the nylon membrane. This is termed slot or dot blotting and provides a convenient means of measuring the abundance of specific mRNA transcripts without the need for gel electrophoresis; it does not, however, provide information regarding the size of the fragments.
A further method of RNA analysis that overcomes the problems of RNA blotting is termed the ribonuclease protection assay. Here the RNA from a sample is extracted and then mixed with a probe representing the sequence of interest in solution. The probe and the appropriate RNA fragment hybridise to form a double-stranded sequence.
RNase is then added, which cleaves any single-stranded RNA present but leaves the double-stranded RNA intact. The intact RNA can then be separated by electrophoresis and an indication of the size of the fragment generated. The efficient removal of the background of RNA and the improved sensitivity make the ribonuclease protection assay a popular choice for the analysis of specific RNA molecules. An important step in the field of RNA analysis was the development of RNAi (RNA interference), which inhibits gene expression. Here double-stranded DNA promotes the degradation of mRNA. Doublestranded RNA in the cell is cleaved by a dicer enzyme, resulting in the
formation of small 21–25 bp interfering RNAs (siRNA). The siRNA are complementary to a target RNA strand. Small RNAi proteins are guided by the siRNA to the appropriate mRNA, where the target is then cleaved and is unable to be translated. Many areas are now benefiting from the adoption of this technique in the molecular biology and biotechnology fields.