title: Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells.
abstract: A method based on the multiplex polymerase chain reaction (PCR) and gel electrophoresis for the comparative analysis of gene expression levels was developed. Using the method many cDNA fragments from different sources can be compared simultaneously. Competitive PCR amplification of expressed genes from different sources was performed by using 'module-shuffling primers' (MPSs). The MPSs (labeled with different fluorophores) consist of sequence modules of 3 or 4 nt. The modules are arranged in different orders in each primer; therefore, the base sequences of the primers are different but their melting temperatures are identical. The genes expressed in different sources are ligated with tags complementary with the MPSs. Tag-ligated fragments are mixed in one tube and amplified at the same amplification efficiency by the MPSs. Amplified fragments are detected separately by multiple-color gel electrophoresis. This method can detect different amounts of each expressed gene, up to a difference in amounts of 30%, and its detection limit is 0.1 amol per assay.
authors.affiliation: Central Research Laboratory, Hitachi Limited, 1-280 Higashi-Koigakubo, Kokubunji, Tokyo 185-8601, Japan.
authors.forename: C
authors.initials: C
authors.lastname: Uematsu
authors.forename: J
authors.initials: J
authors.lastname: Nishida
authors.forename: K
authors.initials: K
authors.lastname: Okano
authors.forename: F
authors.initials: F
authors.lastname: Miura
authors.forename: T
authors.initials: T
authors.lastname: Ito
authors.forename: Y
authors.initials: Y
authors.lastname: Sakaki
authors.forename: H
authors.initials: H
authors.lastname: Kambara
mesh_terms: D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002138:Calibration; D002460:Cell Line; D003116:Color; D017931:DNA Primers / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D005914:Globins / Q000235:genetics; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D013816:Thermodynamics
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D005914:Globins