Quantification of CCR5 mRNA in human lymphocytes and macrophages by real-time reverse transcriptase PCR assay.
CCR5, a beta-chemokine receptor, plays an important role in human immunodeficiency virus (HIV) infection of human immune cells, as it is a primary coreceptor for HIV entry into macrophages. We have applied a newly developed real-time reverse transcriptase PCR (RT-PCR) assay for the quantification of CCR5 mRNA in human blood immune cells. The CCR5 real-time RT-PCR assay has a sensitivity of 100 mRNA copies, with a dynamic range of detection between 10(2) and 10(6) copies of the CCR5 mRNA transcripts per reaction. The assay is highly reproducible, with an intra-assay coefficient of variation of the threshold cycle of less than 2.07%. When used for quantification of CCR5 mRNA in human monocyte-derived macrophages (MDM) and peripheral blood lymphocytes (PBL), the assay has precision and reproducibility. MDM expressed higher levels of CCR5 mRNA than did PBL. Thus, this assay has the potential and a wide application for the investigation of the role of CCR5 in inflammatory diseases and viral infections, including HIV disease.
Lai
Jian-Ping
JP
Divisions of Immunologic and Infectious Diseases, Joseph Stokes, Jr., Research Institute, The Children's Hospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Yang
Ji-Hong
JH
Douglas
Steven D
SD
Wang
Xu
X
Riedel
Eric
E
Ho
Wen-Zhe
WZ
D001773:Blood Cells; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry*; D008264:Macrophages / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*; D019713:Receptors, CCR5 / Q000096:biosynthesis / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D019713:Receptors, CCR5