title: High-throughput RT-PCR analysis of multiple transcripts using a microplate RNA isolation procedure.
abstract: We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR product visualization of a multiwelled agarose gel, stained with SYBR Green I dye. The transcriptional assay was unaffected by solvents (dimethyl sulfoxide and methanol) routinely used in high-throughput drug screens at concentrations required for compound solubilization. Furthermore, it has been used successfully for the investigation of differential mRNA expression levels of tumor necrosis factor alpha (TNF-alpha) and Interleukin-1 beta (IL-1 beta) in lipopolysaccharide (LPS)-stimulated THP-1 cells (a human monocytic cell line) and the identification of specific IL-1 beta transcriptional inhibitors.
authors.affiliation: Glaxo Wellcome Research and Development, Stevenage, Herts, UK.
authors.forename: S
authors.initials: S
authors.lastname: Su
authors.forename: R G
authors.initials: RG
authors.lastname: Vivier
authors.forename: M C
authors.initials: MC
authors.lastname: Dickson
authors.forename: N
authors.initials: N
authors.lastname: Thomas
authors.forename: M K
authors.initials: MK
authors.lastname: Kendrick
authors.forename: N M
authors.initials: NM
authors.lastname: Williamson
authors.forename: J G
authors.initials: JG
authors.lastname: Anson
authors.forename: J G
authors.initials: JG
authors.lastname: Houston
authors.forename: F F
authors.initials: FF
authors.lastname: Craig
mesh_terms: D052160:Benzothiazoles; D002460:Cell Line; D017931:DNA Primers; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007375:Interleukin-1 / Q000235:genetics; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D018942:Macrolides; D009000:Monocytes / Q000378:metabolism; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D012997:Solvents; D013844:Thiazoles / Q000494:pharmacology; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D007093:Imidazoles; D007375:Interleukin-1; D007783:Lactones; D008070:Lipopolysaccharides; D018942:Macrolides; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; D012997:Solvents; D013844:Thiazoles; D014409:Tumor Necrosis Factor-alpha; C098022:SYBR Green I; C052026:6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridinyl)imidazo(2,1-b)thiazole; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C035359:monorden