Quantitation of changes in P0 mRNA by polymerase chain reaction in primary cultured Schwann cells stimulated by axolemma-enriched fraction.
A requirement for large numbers of primary culture cells has frequently restricted investigations of gene expression in glial cells. We have developed a non-radioactive method based on reverse transcription-polymerase chain reaction (RT-PCR) to accurately assess small changes in the expression of the myelin specific gene P0 in Schwann cells. Using axolemma-enriched fraction (AEF) as an inducing agent, we demonstrate that RT-PCR can be used to detect 4-8-fold increases in P0 mRNA levels occurring in a time and dose dependent manner, utilizing only 250000 cells per assay. Initial experiments used an in vitro transcribed RNA for P0 constructed with a 300 bp deletion for quantitation by competitive RT-PCR. Relative quantitation by co-amplification of the housekeeping gene glyceraldehyde-phosphate dehydrogenase was established and provided similar results. Product evaluation was enhanced 50-100-fold by the incorporation of primers labelled with biotin at the 5' end, allowing for the sensitive detection of PCR product by enhanced chemiluminescence and autoradiography. This technique provides sensitivity to detect and evaluate picogram amounts of DNA. Our results validate the assay for P0 gene expression and indicate that the technique should facilitate the study of multiple genes of interest in glial cell systems.
Research Service, Edward Hines Jr Department of Veterans Affairs Hospital, Hines, IL 60141, USA.
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D000818:Animals; D001933:Brain Stem / Q000166:cytology; D002417:Cattle; D002452:Cell Count; D002478:Cells, Cultured; D004996:Ethidium / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000235:genetics; D008163:Luminescent Measurements; D008274:Magnesium / Q000737:chemistry; D018993:Myelin P0 Protein / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012583:Schwann Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012584:Sciatic Nerve / Q000166:cytology; D012680:Sensitivity and Specificity; D013268:Stimulation, Chemical; D013347:Subcellular Fractions / Q000502:physiology; D015854:Up-Regulation
D018993:Myelin P0 Protein; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D004996:Ethidium; D008274:Magnesium