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The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers
Published on: January 22, 2013
[Construction and screening of suppression subtractive hybridization library of renal cell carcinoma]
Yong Zhang1, Jun-kui Ai, Zhi-wen Zhang
1Institute of Urology, First Hospital, Peking University, Beijing 100034, China.
Objective:
To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC).
Methods:
Poly A(+) RNA was isolated from RCC lines 786-O (tester) and renal cell (RC) lines HK-2 (driver), respectively. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit (Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F'. All positive clones picked out were digested and some of which were sequenced.
Results:
The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly, 2 represented unknown genes and the other 48 derived from 36 known genes.
Conclusion:
The quality of the SSH library of human RCC is reliable and its construction is the basis for further screening differentially expressed genes of RCC.
