| 18226543 |
Sauder C, Pedras-Vasconcelos J, Puig M, Verthelyi D: 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. Cytokine. 2008 Feb;41(2):182-6. Epub 2008 Jan 15.
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. |
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