Rat porphobilinogen deaminase gene: a pseudogene-free internal standard for laser-assisted cell picking.
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.
Fink
L
L
University of Giessen, Germany.
Stahl
U
U
Ermert
L
L
Kummer
W
W
Seeger
W
W
Bohle
R M
RM
D000818:Animals; D001483:Base Sequence; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005629:Frozen Sections; D011163:Hydroxymethylbilane Synthase / Q000235:genetics*; D007834:Lasers; D008297:Male; D008969:Molecular Sequence Data; D011544:Pseudogenes; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012333:RNA, Messenger; D011163:Hydroxymethylbilane Synthase