Rat porphobilinogen deaminase gene: a pseudogene-free internal standard for laser-assisted cell picking

Biotechniques. 1999 Mar;26(3):510-6. doi: 10.2144/99263rr02.

Abstract

Analysis of gene expression and its transcriptional regulation requires a reliable access to target mRNA. However, mRNA extractions from homogenized tissue are limited because only average data are obtained, and cell-specific expression may not be addressed. Here, we describe a new method that combines the microscopic selection of oligocellular clusters or a few isotypic cell profiles from complex tissues by UV-laser-assisted cell picking with a simplified and highly efficient protocol for mRNA amplification. For positive control and quantitation reference, a reliable housekeeping gene is needed. For this purpose, we designed primers of the rat porphobilinogen deaminase (PBGD) gene. In contrast to many commonly used housekeeping primer pairs that co-amplify processed pseudogenes, this sequence allowed detection of a pseudogene-free rat cDNA sequence, thus eliminating the need for a DNase-digestion step. PBGD mRNA was consistently expressed in all complex tissues investigated and in 21 specific cell types harvested by laser-assisted cell picking. PBGD is suggested as a reliable new rat housekeeping gene, particularly suitable for analysis of oligocellular samples such as those obtained by laser-assisted cell picking in complex tissues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Separation / instrumentation
  • Cell Separation / methods
  • DNA, Complementary / chemistry
  • DNA, Complementary / genetics
  • Frozen Sections
  • Hydroxymethylbilane Synthase / genetics*
  • Lasers
  • Male
  • Molecular Sequence Data
  • Pseudogenes
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification
  • Rats
  • Rats, Inbred Lew
  • Rats, Sprague-Dawley
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • DNA, Complementary
  • RNA, Messenger
  • Hydroxymethylbilane Synthase

Associated data

  • GENBANK/Y17155