Methods for detecting transcribed pseudogenes: PCR on regions of high sequence similarity followed by cloning and...

Identifying pseudogene transcription is problematic in many cases due to the high sequence similarity between pseudogenes and their parental genes. In this chapter, we detail the procedure for the detection of pseudogene transcription using the reverse transcription polymerase chain reaction (RT-PCR) method. The protocol comprises (1) extraction of total RNA, (2) first-strand cDNA synthesis from total RNA, (3) amplification of the cDNA by PCR, and (4) cloning and sequencing of the PCR products. Technical and practical guidance is provided, and the critical points during each of the steps are discussed. In particular, the importance of designing high specific PCR primers and thoroughly eliminating genomic DNA contamination from RNA preparation is emphasized.