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mCK - 5c多探针核糖核酸酶保护分析试剂盒的IP10(CXCL10)特异性cDNA探针带有两个核苷酸插入,这使得结果的解读变得复杂。

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.

作者信息

Sauder Christian, Pedras-Vasconcelos Joao, Puig Montserrat, Verthelyi Daniela

机构信息

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.

出版信息

Cytokine. 2008 Feb;41(2):182-6. doi: 10.1016/j.cyto.2007.11.009. Epub 2008 Jan 15.

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.

摘要

采用多探针组的核糖核酸酶保护分析(RPA)是同时测量几种不同基因转录的强大工具。我们使用BD生物科学公司/Pharmingen公司的小鼠趋化因子探针组mCK-5c来测量小鼠脑和脾组织中趋化因子基因的表达。根据所使用的RPA方案,我们观察到干扰素诱导蛋白10(IP-10)和T细胞激活-3(TCA-3)转录本的相对量存在差异。从mCK-5c探针组中分离并测序IP-10特异性基因,发现在探针中有两个核苷酸插入,而天然IP-10 cDNA中不存在这些插入。我们表明,这些插入导致受保护的IP-10 mRNA发生核糖核酸酶A依赖性降解,产生一个大小与TCA-3特异性片段无法区分的片段,从而导致对TCA-3表达的过度解读以及对IP-10基因表达水平的低估。

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