Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue.
One of the key end points for understanding the molecular basis of carcinogenesis is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct cells for molecular analysis. A recent advance in microdissection uses the PixCell laser capture microdissection (LCM) system, which allows for precise removal of pure cell populations from morphologically preserved tissue sections. The objective of this study was to determine the optimal fixation protocol for analyzing RNA from tissue samples using LCM. Optimal fixation must provide acceptable morphology, allow proper laser capture of selected cells, and preserve the integrity of mRNA. We evaluated the effects of both cross-linking and precipitive-type fixatives on frozen and paraffin-embedded mouse liver tissue. For assessment of the quality of the mRNA in LCM samples generated from various fixed tissues, reverse transcription-polymerase chain reaction (RT-PCR)-amplified mouse liver beta2-microglobulin mRNA was detected with ethidium bromide. We also examined mouse glyceraldehyde-3-phosphate-dehydrogenase by using the fluorogenic TaqMan system for real-time quantitative detection of RT-PCR products. Frozen tissues yielded more RT-PCR product than did paraffin-embedded tissues. In both frozen and paraffin-embedded tissues, differences were observed between the fixatives. Precipitive fixatives, such as ethanol and acetone, consistently produced more RT-PCR amplification product than did cross-linking fixatives such as formalin. Optimal fixation protocols for LCM analysis will facilitate the examination of gene expression in specific cell populations, accelerating investigations of the molecular differences responsible for the phenotypic changes observed during carcinogenesis.
Goldsworthy
S M
SM
Pathology Associates International, Durham, North Carolina, USA.
Stockton
P S
PS
Trempus
C S
CS
Foley
J F
JF
Maronpot
R R
RR
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005404:Fixatives; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D007834:Lasers; D008099:Liver / Q000378:metabolism; D051379:Mice; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001613:beta 2-Microglobulin / Q000235:genetics
D017931:DNA Primers; D005404:Fixatives; D001613:beta 2-Microglobulin; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases