Luckow B, Maier H, Chilla S, Pérez de Lema G
Medizinische Poliklinik, Arbeitsgruppe Klinische Biochemie, Ludwig-Maximilians-Universität, Schillerstrabetae 42, München, D-80336, Germany.
Anal Biochem. 2000 Nov 15;286(2):193-7. doi: 10.1006/abio.2000.4809.
The ribonuclease protection assay (RPA) represents a sensitive method to detect and quantify RNA levels. It can be adapted to allow the simultaneous analysis of more than 10 different mRNAs. The multiprobe RPA kit mCK-5 from PharMingen was used to analyze the expression of chemokines in CCR5 chemokine receptor knockout mice. Upon careful analysis it was found that the mCK-5 kit is defective and can lead to false results for the chemokines IP-10 and MCP-1. The problem is caused by a long-known sequence polymorphism within the 3'-untranslated region of the murine IP-10 gene. This polymorphism leads to a protected IP-10 fragment approximately 20 nucleotides shorter than expected, yielding a length similar to the protected MCP-1 fragment from the mCK-5 kit. Since the identification of specific transcripts with this kit is based exclusively on the size of the various protected fragments, false-negative results for IP-10 together with false-positive results for MCP-1 can be obtained. Interestingly, the polymorphism was found not only in 129/CD-1 mice, but also in MRL and SJL/J mice. To facilitate troubleshooting in the future, all templates from the mCK-5 set were isolated and sequenced.
核糖核酸酶保护分析（RPA）是一种检测和定量RNA水平的灵敏方法。它可用于同时分析10多种不同的mRNA。使用PharMingen公司的多探针RPA试剂盒mCK-5分析CCR5趋化因子受体基因敲除小鼠中趋化因子的表达。经过仔细分析发现，mCK-5试剂盒存在缺陷，可能导致趋化因子IP-10和MCP-1的检测结果出现错误。该问题是由鼠类IP-10基因3'-非翻译区内一个早已为人所知的序列多态性引起的。这种多态性会导致一个受保护的IP-10片段比预期短约20个核苷酸，其长度与mCK-5试剂盒中受保护的MCP-1片段相似。由于使用该试剂盒鉴定特定转录本完全基于各种受保护片段的大小，因此可能会得到IP-10的假阴性结果以及MCP-1的假阳性结果。有趣的是，这种多态性不仅在129/CD-1小鼠中存在，在MRL和SJL/J小鼠中也存在。为便于今后排除故障，对mCK-5试剂盒中的所有模板进行了分离和测序。
