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首页> 外文期刊>Cytokine >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.
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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.

机译:mCK-5c多探针RNase保护测定试剂盒的IP10(CXCL10)特异性cDNA探针带有两个核苷酸插入片段,使结果的解释复杂化。

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摘要

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.
机译:采用多探针集的RNase保护测定(RPA)是强大的工具,可以同时测量几种不同基因的转录。我们使用了BD Biosciences / Pharmingen的小鼠趋化因子探针集mCK-5c来测量小鼠脑和脾组织中的趋化因子基因表达。根据所使用的RPA协议,我们观察到干扰素诱导蛋白10(IP-10)和T细胞活化3(TCA-3)的相对转录本数量不同。从mCK-5c探针组分离和测序IP-10特定基因后,发现探针中的两个核苷酸插入不存在于天然IP-10 cDNA中。我们显示,这些插入会导致受保护的IP-10 mRNA的RNase A依赖性降解,从而产生与TCA-3特异的大小无法区分的片段,从而导致对TCA-3表达的过度解释以及对IP--3的低估10个基因表达水平。

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