Analysis of RNA by Northern Blotting Using Riboprobes

In studying the expression of a gene in mammalian tissues, it is important to determine the levels of the corresponding mRNA. Several methods have been described for measuring the level of expression of a gene in a tissue. These methods are in situ hybridization using cDNA probe or riboprobe (1 ), slot-blot hybridization on total RNA isolated from tissues (2 ), and Northern blotting hybridization using either a cDNA (3 ) or a riboprobe (4 ). Absolute levels of a specific mRNA are also determined by RNase protection assay using cDNA probe (5 ) or a riboprobe (6 ). In situ hybridization provides relative expression of a gene, whereas the slot-blot technique is not sensitive enough for accurate measurements of mRNA. The most widely used technique to measure mRNA levels in mammalian tissues is still Northern blotting analysis, in which total RNA or poly(A⁺ ) RNA is separated by electrophoresis in an agarose gel-containing formaldehyde and transferred onto a nitrocellulose or nylon membrane. The transferred RNA on the membrane is then denatured and probed with a labeled cDNA probe or a riboprobe. Although the cDNA probe gives the same information as the riboprobe, the sensitivity of the detection of mRNA is increased severalfold by using a riboprobe (4 , and Fig. 1 ) because of the increased affinity of riboprobe for the complementary sense strand of mRNA and higher stability of the double-stranded RNA after hybridization. However, the sensitivity of various membranes may differ marginally while a riboprobe is used (4 ). Thus, the higher sensitivity of the riboprobes in Northern blotting analysis makes them a better choice over cDNA probes especially when detecting a low-abundance message. However, care must be taken while using riboprobes for Northern blotting analysis, as improper use of riboprobe may result in a very high background often by irreversibly binding the riboprobe to the membrane. Therefore, the steps shown in this chapter should be strictly followed in order to avoid background noise.