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.
Luckow
B
B
Medizinische Poliklinik, Arbeitsgruppe Klinische Biochemie, Ludwig-Maximilians-Universität, Schillerstrabetae 42, München, D-80336, Germany. luckow@medpoli.med.unimuenchen.de
Maier
H
H
Chilla
S
S
Pérez de Lema
G
G
D000818:Animals; D001483:Base Sequence; D002623:Chemistry Techniques, Analytical / Q000379:methods; D018932:Chemokine CCL2 / Q000235:genetics*; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000235:genetics*; D004247:DNA / Q000235:genetics; D051379:Mice; D019463:Mice, Inbred MRL lpr; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D019713:Receptors, CCR5 / Q000172:deficiency / Q000235:genetics; D012260:Ribonucleases; D012689:Sequence Homology, Nucleic Acid
D018932:Chemokine CCL2; D054357:Chemokine CXCL10; D019743:Chemokines, CXC; D012333:RNA, Messenger; D019713:Receptors, CCR5; D004247:DNA; D012260:Ribonucleases