An optimized protocol for mRNA quantification using nested competitive RT-PCR.

Abstract

Nested competitive reverse transcription polymerase chain reaction is an effective tool for the quantification of low level expressed mRNAs. Competitive RNA standards with small deletions allow the use cRT-PCR. The sensitivity is further increased by the utilization of nested PCR protocols. To optimize quantification of low abundance mRNAs we modified established protocols for use of automated labstations and semiautomated sequencers. In placental tissue, known for a very high CYP19 (P450AROM, aromatase) expression, cRT-PCR and nested cRT-PCR yielded virtually identical results which could be confirmed by Northern blotting. CYP19 mRNA in breast tumour tissue ranged below detection levels for Northern blotting; however, using our modified assay CYP19 showed 1.5 to 15 fold higher expression levels than in normal glandular breast tissue. Our approach proved to be useful for the quantification of a gene with low level expression. The employment of labstations and semiautomated sequencers allows rapid analysis of large sample numbers.