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 analysi s or RNase protection assays for quantitation of gene expression. To q uantify different samples, measurements are often normalized using the expression of so-called ''housekeeping'' genes, such as cytoplasmic b eta-actin or glyceraldehyde-3-phosphate dehydrogenase. This approach c an produce false results because the presence of processed pseudogenes in the genome, which are related to some of the commonly used transcr ipts of housekeeping genes, leans to coamplification of contaminating genomic DNA. By yielding amplification products of the same or similar size as the reverse-transcribed target, mRNA quantitation of expressi on is prone to error. In this paper, we report the results of using th ree sets of beta-actin primers for RT-PCR in the presence and absence of genomic DNA. In addition, we propose two new parts of oligonucleoti de primers that specifically amplify the human and rat beta-actin reve rse-transcribed mRNA but not pseudogene sequences. These primers are e specially suitable for quantitation of mRNA in small tissue samples (e .g., biopsies), where DNase digestion is not feasible, and therefore D NA contamination cannot be avoided.