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[Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia]
Lei Ji1, Wang-Gang Zhang, Jie Liu
1Department of Hematology, The Second Hospital, Xi'an Jiaotong University, Xi'an 710004, China.
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.
Insights
This study constructed a high-quality subtractive cDNA library to identify genes involved in multidrug resistance (MDR) in acute leukemia. The method efficiently screens for differential gene expression, aiding leukemia treatment research.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Context:
- Acute leukemia is a significant health concern, often complicated by multidrug resistance (MDR).
- Identifying genes responsible for MDR is crucial for developing effective therapeutic strategies.
- Existing methods for gene discovery can be time-consuming and require substantial sample amounts.
Purpose:
- To construct a subtractive cDNA library specifically for identifying genes associated with multidrug resistance (MDR) in acute leukemia.
- To optimize subtractive hybridization and PCR techniques for efficient gene screening.
Summary:
- An improved PCR-based subtractive hybridization method was employed to create a cDNA library comparing HL-60/VCR (MDR) and HL-60 (sensitive) leukemia cell lines.
- Differential gene fragments were cloned, and the library's quality, characterized by a high proportion of large fragments, was confirmed via dot-blotting hybridization.
- This approach enables rapid screening of numerous candidate fragments from minimal samples.
Impact:
- Successfully established a high-quality, large-fragment subtractive cDNA library for MDR in leukemia.
- Provides a valuable tool for efficiently screening differentially expressed genes in leukemia MDR.
- Facilitates further research into the genetic mechanisms underlying leukemia drug resistance and potential therapeutic targets.