[Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia].
The study was aimed to construct subtractive cDNA library associated with multidrug resistance (MDR) of acute leukemia for screening genes related to MDR in leukemia. The improved PCR-based subtractive hybridization was performed to clone differential genes between HL-60/VCR and HL-60 cell line. The mRNA of HL-60/VCR and HL-60 cell line were isolated. Then the mRNA of HL-60/VCR group was reversely transcribed into cDNA by Cap-Finder method, and the mRNA of HL-60 was reversely transcribed into cDNA by ordinary method to be marked by biotin for the hybridization next with HL-60/VCR cDNA. After hybridizing, filtrating through the sephacryl S-400 column, absorbing by the magnetic beads, and amplifying by PCR method, the fragments were cloned by T-A method and the cDNA library was constructed. Then the quality of cDNA library was identified by dot-blotting hybridization method. The results showed that after constriction, the library demonstrated its good quality. There was a high proportion of large fragments in this library. From small amount of samples a large amount of candidate fragments could be screened rapidly at once by dot-blotting hybridization. It is concluded that a differentially-expressed subtractive cDNA library in MDR of leukemia with high quality and larger fragments can be efficiently constructed by improving subtractive hybridization and selective PCR method.
Ji
Lei
L
Department of Hematology, The Second Hospital, Xi'an Jiaotong University, Xi'an 710004, China.
Zhang
Wang-Gang
WG
Liu
Jie
J
Liu
Xin-Ping
XP
Yao
Li-Bo
LB
D000208:Acute Disease; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D015723:Gene Library; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*