Analysis of RNA by Northern Blotting Using Riboprobes

To study 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 (), slotblot hybridization on total RNA isolated from tissues (), and Northern blotting hybridization using either a cDNA () or a riboprobe (). Absolute levels of a specific mRNA are also determined by RNase protection assay using a cDNA probe () or a rrboprobe (). In situ hybridization provides relative expression of a gene, while 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 analysrs, in which total RNA or poly(A+) RNA are separated by electrophoresrs in an agarose gel containing formaldehyde and transferred onto mtrocellulose or nylon membrane. The transferred RNAs on the membrane are then denatured and probed with a labeled cDNA probe or a riboprobe. While a cDNA probe gives the same mformation as the riboprobe, the sensitivity of the detection of mRNA is increased several fold by using a riboprobe (ref. and Fig. 1) because of the increased affinity of riboprobe for the complementary sense strand of mRNA and the higher stability of the double-stranded RNA after hybridization. However, the sensitivity of various membranes may marginally differ while using a riboprobe (). 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 since improper use of riboprobes may result in very high background, often by irreversible binding of the riboprobe to the membrane. Therefore, the steps shown in this chapter should be followed strictly in order to avoid background noise.