Abstract:

BACKGROUND: At present, the most reliable and commonly used methods are guanidine isothiocyanate based RNA isolation method (typically Trizol) and the silica-gel column based RNA isolation method. In case of clinical rare tissues, it is necessary to evaluate the reliability and repetitiveness of the optional RNA isolation methods.
OBJECTIVE: To compare the silica-gel column based RNA extraction method and Trizol method to determine which technique is preferable when frozen, long-term stored or fresh gastric caricinoma tissues have to be evaluated for the downstream molecular analysis.
METHODS: Frozen gastric carcinoma tissues were prepared by long-term storage (more than 2 years) in -80 ℃ while fresh tissues were harvested and processed immediately. The purity of RNA was determined with a spectrophotometer whereas the Ct values of target sequences were recorded by quantitative reverse transcription-PCR (qRT-PCR) system to indirectly assess the RNA intactness (28S and 18S) and mRNA fragment sizes.
RESULTS AND CONCLUSION: The purity of RNA extracted with silica-gel column based and Trizol method was comparable. qRT-PCR data revealed that lower mean Ct values of 28 S and a longer fragment were obtained from RNA isolated with silica-gel column based method than Trizol method using fresh as well as frozen tissues. A low mean Ct value of 18 S and medium fragment were obtained in RNA isolated by silica-gel column based method from the fresh tissues, only. For the shorter fragment, no significant differences were observed. Silica-gel column based RNA isolation method was much more superior to typical Trizol method with respect to the reliable generation of an intact RNA and effective amplification of longer mRNA products in fresh as well as in frozen gastric carcinoma tissues.