Pseudogene-free amplification of HPRT1 in quantitative reverse transcriptase polymerase chain reaction

Anal Biochem. 2015 Sep 15:485:46-8. doi: 10.1016/j.ab.2015.05.021. Epub 2015 Jun 4.

Abstract

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) provides a powerful tool for precise gene expression analysis. The accuracy of the results highly depends on careful selection of a reference gene for data normalization. HPRT1 (hypoxanthine phosphoribosyl transferase 1) is a frequently used housekeeping gene for normalizing relative expression values. However, the existence of processed pseudogenes for HPRT1 might interfere with reliable results obtained in qRT-PCR due to amplification of unintended products. Here, we designed a primer pair for pseudogene-free amplification of HPRT1 in qRT-PCR. We demonstrate that this primer pair specifically amplified HPRT1 messenger RNA (mRNA) sequence while avoiding coamplification of the pseudogenes.

Keywords: HPRT1; Pseudogenes; Reference gene; qRT-PCR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • DNA Primers / chemistry*
  • DNA Primers / genetics*
  • Humans
  • Hypoxanthine Phosphoribosyltransferase / chemistry
  • Hypoxanthine Phosphoribosyltransferase / genetics*
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*

Substances

  • DNA Primers
  • RNA, Messenger
  • Hypoxanthine Phosphoribosyltransferase