Construction of the subtracted cDNA library for HL-60 cells in leukemia G2/M arrest induced by DADS and its preliminary screening.

Objective:To construct a subtracted cDNA library for the HL-60 cells in leukemia G2/M arrest induced by DADS,and to investigate the expression in HL-60 cells G2/M arrest.Methods:Total RNA and mRNA were isolated from HL-60 cells treated with and without DADS,respectively.The suppressive subtractive cDNA library was builded by suppressive subtractive hybridization(SSH). The patterns of the differentially expressing-gene fragments were analyzed by PCR.Sequences of cDNA fragments were compared with that of genes in GenBank by blastn.Results:Subtracted cDNA library for the HL-60 cells in leukemia G2/M arrest induced by DADS was successfully constructed,which contains 120 positive directional clones and 100 negative directional clones.Random analysis of 50 positive directional clones with PCR amplification showed that most clones contained inserted fragments.5 differential fragments were shown to be novel EST and were registered in GenBank.Conclusion:It is evident that subtracted cDNA library for the HL-60 cells in leukemia G2/M arrest induced by DADS was successfully constructed,which provided solid foundation to screen the related genes involved in the HL-60 cells cell cycle arrested in G2/M.