title: Quantification of iNOS mRNA with reverse transcription polymerase chain reaction directly from cell lysates.
abstract: Inducible nitric oxide synthase (iNOS) is a member of a family of primary inflammatory response genes. Quantitative measurement of iNOS mRNA levels is important for the study of gene expression of this enzyme during the process of inflammation. We report here a method for quantitative measurement of iNOS mRNA levels with rtPCR directly from cells lysed with a single step phenol/chloroform/ether extraction. Using a mouse macrophage cell line, J774.2, which expresses iNOS mRNA upon LPS + IFN-gamma treatment as the model, the effects of the extraction on iNOS mRNA recovery and cytosolic RNase removal have been studied. The cells are lysed and RNases denatured and removed by phenol/chloroform extraction. Trace amounts of the phenol partitioned in the samples are then removed by ether extraction. After the extraction, the samples can be used directly for reverse transcription and PCR without further purification of RNA. The recovery of specific mRNA is not affected by the extraction procedure and externally added iNOS cRNA shows no degradation by the extracted cell lysates. Measurement of iNOS mRNA with this procedure is linear using serially double-diluted cells in the range from 94 to 6000 cells. The efficiencies of rtPCR of iNOS wild-type and deletion cRNAs are also compared in our study. By controlling the molecular size of the deletion construct to within 10% of that of the wild type and maintaining PCR cycling below 25 cycles, the rtPCR efficiencies of iNOS wild type and deletion are identical. The detection of rtPCR products is enhanced by hybridization with specific probes. Under these conditions, iNOS mRNA concentration can directly be calculated from the internal standard in each tube without a standard curve. We conclude that our procedure provides an accurate method for quantitative measurement of iNOS mRNA from limited amount of cells without complete RNA isolation.
authors.affiliation: Department of Biological Sciences, State University of New York at Buffalo, Amherst, New York 14260, USA.
authors.forename: B
authors.initials: B
authors.lastname: Han
authors.forename: D C
authors.initials: DC
authors.lastname: DuBois
authors.forename: K M
authors.initials: KM
authors.lastname: Boje
authors.forename: S J
authors.initials: SJ
authors.lastname: Free
authors.forename: R R
authors.initials: RR
authors.lastname: Almon
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D004790:Enzyme Induction / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D018075:RNA, Complementary / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D008070:Lipopolysaccharides; D018075:RNA, Complementary; D012333:RNA, Messenger; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse