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Anal Biochem. 2000 Nov 15;286(2):193-7.

The mCK-5 multiprobe RNase protection assay kit can yield erroneous results for the murine chemokines IP-10 and MCP-1.

Author information

1
Medizinische Poliklinik, Arbeitsgruppe Klinische Biochemie, Ludwig-Maximilians-Universität, Schillerstrabetae 42, München, D-80336, Germany. luckow@medpoli.med.unimuenchen.de

Abstract

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.

PMID:
11067740
DOI:
10.1006/abio.2000.4809
[Indexed for MEDLINE]

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