The mCK-5 multiprobe RNase protection assay kit can yield erroneous results for the murine chemokines IP-10 and MCP-1.
Anal Biochem
; 286(2): 193-7, 2000 Nov 15.
Article
em En
| MEDLINE
| ID: mdl-11067740
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.
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Base de dados:
MEDLINE
Assunto principal:
Ribonucleases
/
Quimiocina CCL2
/
Quimiocinas CXC
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2000
Tipo de documento:
Article