Quantitative Determination of Gene Expression Level by QPCR Method

DOI 2 Citations 12 References
  • NAKAYAMA Hiroshi
    Department of Biochemistory II, Kyourin University School of Medicine
  • NAGASHIMA Yasushi
    Department of Surgery II, Kyourin University School of Medicine
  • FURUYA Hiroshi
    Department of Internal Medicine III, Kyourin University School of Medicine
  • KUROKI Yoshihiro
    Department of Surgery II, Kyourin University School of Medicine
  • HAYAKAWA Ruriko
    Department of Biochemistory II, Kyourin University School of Medicine
  • MIZUNO Hitoshi
    Department of Biochemistory II, Kyourin University School of Medicine
  • YOSHINAGA Emi
    Department of Biochemistory II, Kyourin University School of Medicine
  • SAKAI Tetsuo
    Department of Biochemistory II, Kyourin University School of Medicine
  • WAKIZAKA Akira
    Department of Biochemistory II, Kyourin University School of Medicine

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Other Title
  • QPCR法による遺伝子発現量の定量に関する基礎的研究

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Experimental procedures were analyzed for the QPCR method by which the amount of cDNA, the product of reverse transcriptase reaction of mRNA, could be determined in a high sensitivity to estimate the expression level of a gene. RNA extracted from mouse liver, was first converted to cDNA by the action of reverse transcriptase. Thus obtained cDNA was further amplified by PCR with a set of primers for p53 gene, one of which was labeled with chemoluminescence inducible material, TBR (tris (2,2-bipyridine) ruthenium (II) chelate), and the other with biotin. The PCR products coupled to streptavidine-conjugated magnetic beads, led into the QPCR device and electrochemical luminescence induced from TBR was measured. In 30 times of the amplification the luminosity was approximately proportional to the amount of RNA between 0.01 to 0.25μg added to the assay mixture. In 20 to 25 times of the PCR cycle, RNA in the range of 0.1 to 1.0 μg was correlated with the luminosity. With this method, a minimum of 1.0×10^<-16> moles of cDNA was able to be determined with 13〜14 % of Coefficient of Variation within 3 hours. DNA contaminated in the RNA material by 5 % apparently exhibited no interference on the chemoluminescence production but by 10 % in the material decreased the luminosity to 90 % of the original one. RNA preparation treated with DNase to eliminate the contaminated DNA strongly decreased the luminosity.

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