Aim: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC).
Methods: Suppression subtractive hybridization(SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression.
Results: By this way, there were about 3-4 X 10(2) clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database.
Conclusion: All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.