[Construction of a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma tissue].
OBJECTIVE
To construct a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma (HCC) tissue.


METHODS
Using the suppression subtractive hybridization (SSH), a novel technique has been described recently. cDNA fragments of missing or low expressing tumor suppressor genes in HCC tissue were isolated using paracancerous normal liver tissue and HCC tissue as targets. Then these cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of E.coli by high voltage electroperforation. One hundred positive bacteria clones were randomly picked and identified using enzyme restriction method.


RESULTS
The amplified library contained more than 4,000 positive bacteria clones. Random analysis of 100 clones with enzyme restriction method showed that all clones contained 200-600 bp inserts.


CONCLUSIONS
A subtracted cDNA library of differentially expressed genes in human normal liver tissue and HCC tissue is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for screening and cloning new and specific missing or low expressing tumor suppressor genes of HCC.
Li
J
J
Department of Hepatobiliary Surgery, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.
Xu
X
X
Han
B
B
Huang
G
G
Qian
G
G
Liang
P
P
Yang
T
T
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms / Q000235:genetics*
D018076:DNA, Complementary