Construction of human stem cell factor's RNA-CRS in quantitative RT-PCR

Academic Article

Abstract

  • A novel method was developed to prepare an Exonuclease III-partially:digesting RNA as a competitive reference standard (RNA-CRS) of human stem cell factor (hSCF) gene in quantitative RT-PCR: complete hSCF cDNA was already amplified from HepG2 cells using RT-PCR and cloned into pGEM-T vector. After the recombinant pGEMSCF was treated with Exonuclease III and S1 nuclease at a favorable condition to make a limited deletion in hSCF cDNA, the recombinant pGEMSCF mimic was constructed successfully and transcribed in vitro to obtain the RNA-CRS. The hSCF RNA-CRS with a 110 bp deletion from base 499 to 608 in hSCF cDNA was identified by DNA sequencing and it is suitable to be used as a reliable RNA-CRS for the quantitation of the transcriptional expression level of recombinant hSCF in eukaryotic cells by quantitative RT-PCR.
  • Authors

    Author List

  • Tan WB; Nie YL; Luo SQ; Guo XS; Cheng GJ; Chen HC; Zhu DE
  • Start Page

  • 318
  • Volume

  • 27
  • Issue

  • 3