![]() 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. 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, the sensitivity of various membranes may differ marginally while a riboprobe is used (4). 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. 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. The transferred RNA on the membrane is then denatured and probed with a labeled cDNA probe or a riboprobe. 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. In situ hybridization provides relative expression of a gene, whereas the slot-blot technique is not sensitive enough for accurate measurements of mRNA. Absolute levels of a specific mRNA are also determined by RNase protection assay using cDNA probe (5) or a riboprobe (6). 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). Several methods have been described for measuring the level of expression of a gene in a tissue. In studying the expression of a gene in mammalian tissues, it is important to determine the levels of the corresponding mRNA. ![]()
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