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.
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.
Sauder
Christian
C
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
Pedras-Vasconcelos
Joao
J
Puig
Montserrat
M
Verthelyi
Daniela
D
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
D054402:Chemokine CCL1; D054357:Chemokine CXCL10; C485433:Cxcl10 protein, mouse; D011933:Reagent Kits, Diagnostic; D012259:Ribonuclease, Pancreatic