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

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.

Identification of new regulatory sequences far upstream of the mouse monocyte chemoattractant protein-1 gene.

We systematically searched for sequences influencing the expression of the mouse monocyte chemoattractant protein-1 (MCP-1) gene (Scya2) by mapping DNase I hypersensitive sites (HS) in the chromatin of mesangial cells in a 40-kb interval around the gene. We found nine HS located between -24 kb and +12.7 kb. Three HS coincided with previously known regulatory sequences (HS-2.4, HS-1.0, and HS-0.2). We tested two of the previously unknown HS located far upstream of Scya2 (HS-19.4 and HS-16.3) in transfection experiments using luciferase reporter constructs and mouse mesangial cells as recipients. In transient transfections, both HS had a moderate effect on basal promoter activity as well as promoter activity stimulated by tumor necrosis factor-alpha. In stable transfection experiments, we found much higher activity. A DNA fragment containing HS-19.4 and HS-16.3 caused a considerable increase in the number of stably integrated luciferase copies. We determined the nucleotide sequence of the 5' flanking region to -28.6 kb. Computer-assisted sequence analysis did not yield evidence of an additional gene. These HS are located within the 5' flanking region of a gene cluster consisting of Scya2 (MCP-1), Scya7 (MCP-3), Scya11 (eotaxin), Scya12 (MCP-5), and Scya8 (MCP-2). This report represents the first comprehensive chromatin analysis of the mouse MCP-1 locus leading to the identification of a complex regulatory region located far upstream of Scya2.