Estimation of relative mRNA content by filter hybridization to a polythymidylate probe.
We have developed an accurate and sensitive method for the quantitation of relative mRNA in RNA samples. This procedure is especially applicable for normalizing numerous RNA samples which are to be analyzed by dot blot hybridization. The method entails hybridization of a polythymidylate probe with RNA bound to filters. The relative hybridization of polythymidylate probe to RNA is proportional to the polyadenylate RNA content of the RNA samples. Hybridization of polythymidylate probe to RNA is not dependent on any cell treatment or growth condition that we have observed, and experimental variation is minimized. Therefore, the relative hybridization to polythymidylate probe is a better method for standardizing the amount of mRNA in RNA samples than is relative hybridization to cDNA probes such as actin or beta 2-microglobulin, whose transcript levels may vary according to cell treatment. Furthermore, since experimental variation and errors are minimized, this procedure is suitable for normalizing RNA samples in which there are only small changes in the levels of particular transcripts.
Laboratory of Molecular Pharmacology N.C.I., N.I.H., Bethesda, MD 20892.
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Hollander
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Fornace
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005821:Genetic Techniques; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011071:Poly T; D011089:Polydeoxyribonucleotides; D012333:RNA, Messenger / Q000032:analysis*; D012588:Scintillation Counting
D015345:Oligonucleotide Probes; D011089:Polydeoxyribonucleotides; D012333:RNA, Messenger; D011071:Poly T