title: The IP10 (CXCL10) specific cDNA probe of the mCK-5c multiprobe RNase protection assay kit carries two nucleotide insertions that complicate the interpretation of results.
abstract: RNase protection assays (RPA) employing multiprobe sets are powerful tools to simultaneously measure transcription of several different genes. We used BD Biosciences/Pharmingen's mouse chemokine probeset mCK-5c to measure chemokine gene expression in brain and spleen tissue of mice. Depending on the RPA protocol used, we observed differences in the relative amounts of transcripts for interferon-inducible protein 10 (IP-10) and T-cell activation-3 (TCA-3). Isolation and sequencing of the IP-10 specific gene from the mCK-5c probeset revealed two nucleotide insertions in the probe that are not present in the natural IP-10 cDNA. We show that these insertions cause RNase A-dependent degradation of the protected IP-10 mRNA yielding a fragment indistinguishable in size from that specific for TCA-3, thus leading to over-interpretation of TCA-3 expression as well as underestimation of IP-10 gene expression levels.
authors.affiliation: Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Building 29A, Room 2C-20, HFM460, 8800 Rockville Pike, Bethesda, MD 20892, USA. christian.sauder@fda.hhs.gov
authors.forename: Christian
authors.initials: C
authors.lastname: Sauder
authors.forename: Joao
authors.initials: J
authors.lastname: Pedras-Vasconcelos
authors.forename: Montserrat
authors.initials: M
authors.lastname: Puig
authors.forename: Daniela
authors.initials: D
authors.lastname: Verthelyi
mesh_terms: D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D054402:Chemokine CCL1 / Q000032:analysis*; D054357:Chemokine CXCL10 / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D003951:Diagnostic Errors; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D016415:Sequence Alignment; D013154:Spleen / Q000378:metabolism
chemical_list: D054402:Chemokine CCL1; D054357:Chemokine CXCL10; C485433:Cxcl10 protein, mouse; D011933:Reagent Kits, Diagnostic; D012259:Ribonuclease, Pancreatic