title: Quantitative analysis of multiple genes' expressions based on a novel competitive RT-PCR assay.
abstract: We established a novel gene expression analysis platform, Multiplex Competitive RT-PCR Using Fluorescent Universal Primers (MCF-PCR), to study multi-gene expression patterns simultaneously. This platform combines fluorescent universal primers, multiplex competitive RT-PCR, and capillary electrophoretic separation, which ensures MCF-PCR a reliable, medium-throughput, cost-effective technology for gene expression profiling. With cloned standard DNAs, the detection limits, precision, and sensitivity of MCF-PCR were evaluated and compared with that of the assay without adding competitive templates and real-time PCR, respectively. The results showed that detection limit was 3.125 × 10(3) to 3.2 × 10(6) copies, and 10 % copy differences between two samples can be detected by MCF-PCR. To validate MCF-PCR, we analyzed expression profile of five genes in interleukin (IL)-4/IL-13 pathway in peripheral blood of 20 healthy adults and 20 allergic dermatitis patients; three genes including IL-4, IL-13, and STAT6 were found differentially expressed in the two sample groups, which maybe key players in IL-4/IL-13 immunological signaling pathway and need further function analysis.
authors.affiliation: Department of Laboratory Medicine, Shanghai First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.forename: Li-hui
authors.initials: LH
authors.lastname: Lin
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.forename: Xia
authors.initials: X
authors.lastname: Peng
authors.forename: Ya-nan
authors.initials: YN
authors.lastname: Liu
authors.forename: Jun-hua
authors.initials: JH
authors.lastname: Xiao
authors.forename: Yu-xun
authors.initials: YX
authors.lastname: Zhou
authors.forename: Li
authors.initials: L
authors.lastname: Li
mesh_terms: D000328:Adult; D017931:DNA Primers / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D018793:Interleukin-13 / Q000235:genetics; D015847:Interleukin-4 / Q000235:genetics; D008297:Male; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D050800:STAT6 Transcription Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D018793:Interleukin-13; D050800:STAT6 Transcription Factor; D015847:Interleukin-4