Semiquantitative reverse transcription-polymerase chain reaction with the Agilent 2100 Bioanalyzer.
We have applied a method to monitor mRNA expression in a semiquantitative fashion on the Agilent 2100 Bioanalyzer. The method was originally described in 1994 by Wong et al. and referred to as the "primer-dropping" method. This polymerase chain reaction (PCR) technique uses multiple sets of primer pairs in a coamplification reaction that amplifies the target of interest within a predetermined range specific for each target. Separation, detection and quantification of PCR products were accomplished using the Agilent 2100 Bioanalyzer in conjunction with the DNA 500 and the DNA 1000 Lab-Chip kits for the detection of DNA fragments with a maximum size of 500 and 1000 bp, respectively. Using primers specific for the inducible form of hsp72 and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard we were able to rapidly monitor and quantify inducible hsp72-mRNA expression.
Gottwald
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Forschungszentrum Karlsruhe, Institut fur Medizintechnik und Biopyhsik, Germany. eric.gottwald@imb.fzk.de
Müller
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O
Polten
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A
D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics; D022781:Hepatocytes / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008832:Microchemistry / Q000295:instrumentation*; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation*; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases