Influence of histochemical stains on quantitative gene expression analysis after laser-assisted microdissection.
Laser-assisted microdissection (LAM) allows isolation of specific cell populations for molecular studies. The combination of LAM and of real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) enables generation of quantitative cell-specific gene expression data. Histochemical stains used to identify cells desired for LAM should provide acceptable morphology and not interfere with RNA or with subsequent molecular analysis techniques. To determine a reliable stain for analysing RNA, using the housekeeping gene, RPL13A, we performed quantitative gene expression analysis of laser microdissected cells from prostatic frozen tissues. The frozen sections were histochemically stained with hematoxylin, methyl green, toluidine blue O and May-Grunwald. After laser microdissection real-time quantitative RT-PCR was performed. Methyl green yielded more RT-PCR product than did the other dyes. The lowest yield of amplification was obtained after May-Grunwald staining. Therefore we recommend methyl green for general use in gene expression analysis, especially when handling small amounts of RNA.
Okuducu
Ali-Fuat
AF
Institute of Pathology, University of Bonn, 53011 Bonn, Germany.
Janzen
Viktor
V
Hahne
Jens Claus
JC
Ko
Yon
Y
Wernert
Nicolas
N
D004996:Ethidium / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D011467:Prostate / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000235:genetics*; D013194:Staining and Labeling
C402487:RPL13A protein, human; D012269:Ribosomal Proteins; D012313:RNA; D004996:Ethidium