Pitfalls of processed pseudogenes in RT-PCR.

Pseudogenes are genes with high sequence homology to functional genes that have been rendered obsolete by mutations, preventing their expression. Several genes, such as αand β-globin, have been shown to have duplicated sequences representing nonfunctional genes or pseudogenes. The presence of pseudogenes is common; not every DNA duplication produces a functional gene. Cyclophilin (CyP), an 18-kDa protein originally isolated as the main cyclosporin A-binding protein (1), is a major cellular component, comprising 0.1% to 0.4% of total cellular protein (7). CyP expression is not restricted to a specific cell type but is found in all cells of wide phylogenetic distribution (5,7,9) and might therefore be useful as a housekeeping gene for comparative quantitation of mRNA by reverse transcription polymerase chain reaction (RT-PCR). However, Southern blot analysis of mammalian genomic DNA has revealed multiple CyP-homologous sequences representing related genes or pseudogenes (2,3). Notwithstanding the existence of these pseudogenes, we investigated its use for this purpose with RNA extracted from human peripheral blood mononuclear cells (PBMCs). The cyclophilin primer sequences were obtained from the nucleotide sequence of Haendler and Hoffer (4) and designed to span an intron amplifying a 597-bp fragment. The primer pair sequences used in the amplification process were 3317 (5′ATGGTCAACCCCACCGTGTTCTTCGAC-3′) and 3318 (5′-AATCGAGTTGTCCACAGTCAGCAATGG-3′). Initial results showed the presence of an amplified band of the expected size in the controls where the RNA had not been reverse-transcribed. Upon further investigation of the primer sequence and positioning, we discovered that although the primers were designed across an intron, the DNA sequence and RNA sequence showed high homology, and the above primers would therefore be likely to amplify any DNA contaminating the RNA preparations. We describe a simple method for the preparation of RNA suitable for RT-PCR when RNA-specific primers cannot be designed and that can be used even when pseudogenes are a confounding problem. To minimize the amount of cellular DNA contamination within the RNA preparations, two different methods of RNA extraction were compared. First, total RNA was extracted from 1 × 106 PBMCs isolated from buffy packs using Ficoll-Paque density gradients (Pharmacia Biotech, Uppsala, Sweden) using Total RNA Isolation Reagent (Advanced Biotechnologies, London, England, UK) according to the manufacturer’s protocol. First-strand cDNA synthesis was then performed in a 20μL reaction mixture containing 25 ng