title: Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device.
abstract: This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.
authors.affiliation: School of Science and the Environment, Manchester Metropolitan University, Manchester, UK. k.shaw@mmu.ac.uk
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: Shaw
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Hughes
authors.forename: Charlotte E
authors.initials: CE
authors.lastname: Dyer
authors.forename: John
authors.initials: J
authors.lastname: Greenman
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Haswell
mesh_terms: D000818:Animals; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D022781:Hepatocytes / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008297:Male; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D012313:RNA; D019388:Cytochrome P-450 CYP1A2; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases