Establishing a correlation between RIN and A260/280 along with the multivariate evaluation of factors affecting the quality of RNA in cryopreserved cancer bio-specimen.

Biomarkers are biological characteristic to measure and evaluate normal and pathological states. To define expression-based molecular biomarkers, high-quality tissue samples are a prerequisite for the preparation of standard RNA. It is already known that RIN number defines the RNA quality, however its relation with A260/280 ratio and Ct value is not defined clearly; therefore, understanding an association will provide a reliable method for describing RNA quality. Multiple cryopreserved human tumor tissue types from ACTREC Biorepository and TMH-INTTR were analyzed for the effect of storage time on RNA quality. The RNA from tumor samples were isolated and analyzed by RIN, A260/280 ratio, and Ct value to establish inter-relationships. Around 50% samples had a RIN of ≥ 6.9 and A260/280 ≤ 2.04; 27% had a RIN ≥ 5 and A260/280 ≤ 2.08, and remaining 23% displayed RIN < 5 and A260/280 > 2.08. However, the RNA quality has no association with the storage period of tissue samples. Moreover, all samples which had A260/280 ≤ 2.08 showed acceptable Ct values of 17-24. The data clearly suggests that the A260/280 ratio is able to predict RNA quality. To the best of our knowledge, this is the first Indian report analyzing the labile nucleic acid-RNA quality from different cancer tissue types cryopreserved for diverse time periods. In conclusion, RIN and A260/280 ratio can help in predicting the quality of RNA independently; however, both together provide better assurance for further downstream processing.

Quality assessment of cryopreserved biospecimens reveals presence of intact biomolecules.

Recapitulation of tumor features in isolated biomolecules is preeminently dependent on obtaining reliable quality biospecimen. Moreover, quality assessment of biobanked specimens at regular intervals is an essential intervention for carrying out effective translational and clinical research. In the current study, genomic DNA was extracted from 140 fresh frozen tissues of oral, breast and colorectal specimens cryopreserved over a period of 3 to 8 months (short term) and 3 to 4 years (long term). Quantification of genomic DNA by absorption and fluorescence spectroscopy confirmed high concentration while qualitative analysis by gel electrophoresis showed intact bands for 94% and 87% of short- and long-term cohorts, respectively. PC-LDA based classification of Raman spectra showed overlapping groups of both cohorts suggesting the quality of DNA being preserved irrespective of storage period. To the best of our knowledge this is the first Indian biobank study reporting quality analysis of biospecimens cryopreserved at different time periods.