Design and testing of beta-actin primers for RT-PCR that do not co-amplify processed pseudogenes.
Quantitative reverse transcription polymerase chain reaction (RT-PCR) is being used increasingly as an alternative to Northern blots analysis or RNase protection assays for quantitation of gene expression. To quantify different samples, measurements are often normalized using the expression of so-called "housekeeping" genes, such as cytoplasmic beta-actin or glyceraldehyde-3-phosphate dehydrogenase. This approach can produce false results because the presence of processed pseudogenes in the genome, which are related to some of the commonly used transcripts of housekeeping genes, leads to co-amplification of contaminating genomic DNA. By yielding amplification products of the same or similar size as the reverse-transcribed target, mRNA quantitation of expression is prone to error. In this paper, we report the results of using three sets of beta-actin primers for RT-PCR in the presence and absence of genomic DNA. In addition, we propose two new pairs of oligonucleotide primers that specifically amplify the human and rat beta-actin reverse-transcribed mRNA but not pseudogene sequences. These primers are especially suitable for quantitation of mRNA in small tissue samples (e.g., biopsies), where DNase digestion is not feasible, and therefore DNA contamination cannot be avoided.
Raff
T
T
Universitätsklinikum Benjamin Franklin Free University, Berlin, Germany.
van der Giet
M
M
Endemann
D
D
Wiederholt
T
T
Paul
M
M
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D017931:DNA Primers; D003851:Deoxyribonucleases / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011544:Pseudogenes; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D051381:Rats
D000199:Actins; D017931:DNA Primers; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012194:RNA-Directed DNA Polymerase; D003851:Deoxyribonucleases