[19] Biochemical manipulations of minute quantities of mRNAs and cDNAs immobilized on cellulose paper discs

Publisher Summary This chapter discusses the biochemical manipulations of minute quantities of messenger RNA (mRNAs) and complementary DNA (cDNA) immobilized on cellulose paper discs. In principle, cDNA is prepared directly from small amounts of mRNA trapped on oligo (dT) cellulose paper disks, using the oligo (dT) of the paper as primer for reverse transcription. The newly formed cDNA, covalently linked to the disk, is then employed in further reactions. This procedure: (1) permits manipulations of very small amounts of mRNA and cDNA, (2) reduces the processing time of serial reactions considerably, because the bound nucleic acid may be purified by rinsing in an appropriate buffer rather than by precipitation and centrifugation, and (3) reduces losses of nucleic acid products that might otherwise occur during each of the recovery steps. A target mRNA of interest in a given population of mRNA can be detected or even quantitated by the dot-blot procedure, using an appropriate probe. But the extreme susceptibility of mRNA to degradation by chance contamination with RNase is a practical drawback in the storage and manipulations of RNA samples. However, once reverse transcribed and immobilized on cellulose disk, the cDNA act as stable and durable records of their corresponding mRNAs and can be repeatedly screened by the same or different probes, as needed.