Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia.
- Author:
Lei JI
1
;
Wang-Gang ZHANG
;
Jie LIU
;
Xin-Ping LIU
;
Li-Bo YAO
Author Information
1. Department of Hematology, The Second Hospital, Xi'an Jiaotong University, Xi'an 710004, China.
- Publication Type:Journal Article
- MeSH:
Acute Disease;
Drug Resistance, Multiple;
genetics;
Drug Resistance, Neoplasm;
genetics;
Gene Library;
HL-60 Cells;
Humans;
Leukemia;
drug therapy;
genetics;
Nucleic Acid Hybridization;
methods
- From:
Journal of Experimental Hematology
2004;12(4):431-435
- CountryChina
- Language:Chinese
-
Abstract:
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.
