Highly Sensitive and Specific Fluorescence Reverse Transcription-PCR Assay for the Pseudogene-free Detection of β-Actin Transcripts as Quantitative Reference

Abstract

The use of common reverse transcription (RT)-PCR reference target sequences can produce false-positive results by amplification of either contaminating DNA or processed pseudogenes. Furthermore, qualitative RT-PCR alone cannot distinguish between high- and poor-quality cDNA preparations, which again may be crucial for the interpretation of low-abundance transcripts. We have developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry. Through this technique, we were able to quantitatively detect 10 β-actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA. Thus, the presented method may be advantageous for the interpretation of quantitative RT-PCR results. PCR analysis may be difficult to interpret unless a reference target sequence is amplified in parallel. This control reaction is necessary to evaluate whether a sufficient amount of amplifiable material is present in the sample investigated. Thus, negative PCR can be defined as such only when amplification of a reference gene reveals a positive result. Amplifying a reference is especially important in RT-PCR because RNA can be degraded rapidly before or during cDNA synthesis. As reference sequences for RT-PCR, so called “housekeeping” genes are preferred because they are constitutionally expressed by all cell types. β-Actin is an attractive candidate for reference coamplification because it exhibits only minor intraindividual kinetic changes and is not primarily affected by any human disease (1). However, a major concern with β-actin and other commonly used references such as glyceraldehyde-3-phosphate dehydrogenase is that processed pseudogenes can be coamplified by primers that are originally restricted to cDNA. This problem is difficult to circumvent because sequence homologies of >90% exist between mRNA and the respective pseudogenes (2); thus, the pseudogenes usually cannot be distinguished in gel electrophoresis. Furthermore, as long as genomic DNA contamination cannot safely be excluded, DNA may be coamplified together with cDNA. Thus, positive analysis of …