[Screening of novel genes differentially expressed in human renal cell carcinoma by suppression subtractive hybridization].
OBJECTIVE
Identifying the differentially expressed genes in renal cell carcinoma (RCC) contributes to the elucidation of its genetic basis. However, the above knowledge has not yet been fully understood. The aims of this experiment were to screen novel genes differentially expressed in RCC tissues by suppression subtractive hybridization (SSH) and clone RCC-specific related genes.


METHODS
To construct SSH library of RCC by using the mRNA from RCC tissues and matched normal kidney tissues as tester and driver, respectively. Partial positive clones in the library were selected randomly and sequenced, then analyze the sequences with the BLAST software. To confirm the location of the fragments of interest in human chromosome through comparing their sequences with the human genome draft. mRNA levels of the novel genes in RCC and matched normal kidney tissues were determined by Northern blot and semi-quantitative RT-PCR analysis.


RESULTS
The SSH library contained 414 positive clones. Random analysis of 280 clones with enzyme restriction showed that 265 clones contained cDNA fragments distributed mainly between 300-900bp. Among 80 arbitrary clones with were derived from above 265 clones and sequenced, No. 28, 158, 170, and 249 clones are previously unknown genes and located in human chromosome 21q22, 4p15.3, 9q34, and 22q11.2 by electronic mapping, respectively. The consequence of semi-quantitative RT-PCR demonstrated that mRNA levels of the two novel genes were overexpressed in RCC compared to matched normal tissues by more than 2-6 folds. Northern blot analysis confirmed the above results.


CONCLUSIONS
SSH is a reliable strategy for screening novel genes differentially expressed in RCC. The novel gene fragments can be used to clone their full length and further to study their functions.
Ai
Jun-kui
JK
Institute of Urology, First Hospital, Peking University, Beijing 100034, P. R. China.
Huang
Xiao
X
Wang
Yu-juan
YJ
Bai
Yin
Y
Lu
Ying-qian
YQ
Ye
Xiong-jun
XJ
Xin
Dian-qi
DQ
Na
Yan-qun
YQ
Zhang
Zhi-wen
ZW
Guo
Ying-lu
YL
D015152:Blotting, Northern; D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000473:pathology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger