A practical approach for quantitating specific mRNAs by solution hybridization

✍ Scribed by Diane M. Durnam; Richard D. Palmiter

• Publisher: Elsevier Science
• Year: 1983
• Tongue: English
• Weight: 752 KB
• Volume: 131
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(83)90188-4

✦ Synopsis

The preparation and use of a specific cDNA probe for quantitating mRNA by solution hybridization is described. Cloned DNA sequences are nick translated, denatured, hybridized to single-stranded M 13 clones containing message strand (mDNA) sequences, and separated chromatographically on Bio-Gel A50 under first native and then denaturing conditions to yield a single-stranded cDNA probe. The details of a solution hybridization assay in which the singlestranded cDNA is used to quantitate mRNA in total nucleic acid samples are described. As little as 0.5 pg of mRNA can easily be detected within a day of sample isolation. Thus, the assay is both rapid and sensitive and can be used to measure RNAs complementary to any cloned DNA sequence. It is ideally suited to situations when accurate quantitation of multiple samples is anticipated.