Construction of subtracted cDNA libraries of pancreatic cancer and normal tissue with suppression subtractive hybridization and quality analysis

Objective In an attempt to seek out new factors that are related to pancreatic cancer at the molecular level, suppression subtractive hybridization between cDNA of normal pancreatic tissues and cDNA of pancreatic cancer tissues were performed and subsequent screenings of the subtracted cDNA libraries. Methods The subtracted cDNA libraries were performed using the protocol described in CLONTECH PCR-Select (tm) cDNA Subtraction Kit User Manual. The tester and driver cDNAs were synthesized from 2 μg of poly(A) + RNA from the pancreatic cancer tissues and normal pancreatic tissues being compared, and then digested with Rsa I, a four-base-cutting restriction enzyme that yielded blunt ends. The pancreatic cancer cDNA was then subdivided into two portions, and each was ligated with a different cDNA adaptor. Two hybridizations were then performed. First, an excess of the driver was added to each sample of the tester. During the second hybridization, the two primary hybridization samples were mixed together without denaturing. Using suppression PCR the differentially expressed sequences were significantly enriched. The desired differentially expressed sequences were then inserted into a T/A cloning vector to generate a pancreatic cancer-special subtracted cDNA library. Results Unique pancreatic cancer cDNA libraries with limited normal tissue and tumor from the same patient was constructed via SSH. The amplified library contained 500 positive clones. The fragment amplified by PCR was inserted into plasmid in 457 clones and contained 250-750 bp. Conclusions cDNA library subtraction is a good approach to enrich pancreatic cancer specific changes in gene expression or in gene activation. These changed genes will to be exploited for understanding their role in clinical pancreatic cancer.