Abstract
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.
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Abbreviations
- IDC:
-
Infiltrating ductal carcinoma
- ADC:
-
Adenocarcinoma
- GBM:
-
Glioblastoma
- SN:
-
Seminoma
- ACTREC-TTR:
-
Advanced Centre for Treatment, Research and Education in Cancer-Tumor Tissue Repository
- TMH-INTTR:
-
Tata Memorial Hospital-ICMR National Tumor Tissue Repository
- TNM:
-
T-primary tumor
- N:
-
Node
- M:
-
Metastasis
- DNA:
-
Deoxyribonucleic acid
- RNA:
-
Ribonucleic acid
- RIN:
-
RNA integrity number
- Ct:
-
Cycle threshold
- EMR:
-
Electronic medical record
- DPX:
-
Distyrene, plasticizer, xylene
- qPCR:
-
Quantitative polymerase chain reaction
- RPS13 :
-
Ribosomal protein subunit
- GAPDH :
-
Glyceraldehyde 3-phosphate dehydrogenase
- β-actin :
-
Beta-actin
- S.D.:
-
Standard deviation
- TCGA:
-
The Cancer Genome Atlas
- H and E:
-
Hematoxylin and eosin
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Acknowledgements
Authors are grateful to all members of Gupta Lab, ACTREC for valuable discussions. S.S., M.R. and T.V. are supported by DAE-ACTREC fellowship. This work was supported in part by TMC-Intramural Research Grant. We are thankful to Dr. Manisha Kulkarni and Mr. Anand Deshpande from TMH for their kind co-operation and providing stomach and testes tissue samples on time. The authors are grateful to Dr. Nikhil Patkar, Dr. Shruti and Dr. Sanjeev Waghmare for providing pico-chips.
Funding
This research received intramural grant from ACTREC-TMC (Grant No: IRG#164).
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10561_2019_9782_MOESM1_ESM.tif
Supplementary Fig. 1: RNA integrity number, A260/280 ratio and Ct values of human tumor tissue samples: (a) Correlation between A260/280 ratio and RIN. Tumor samples with A260/280 ratio of ≤ 2.04 represented RIN value ≥ 6.9 displaying a high-quality RNA (dark grey). Tumor samples with A260/280 ratio between 2.05-2.08 and RIN value in-between 5-6.8 depicted a partially degraded RNA (light grey). Tumor samples with A260/280 ratio of > 2.08 and RIN value < 5 displayed a poor-quality RNA (white) (TIFF 7299 kb)
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Shah, S.G., Rashid, M., Verma, T. et al. 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. Cell Tissue Bank 20, 489–499 (2019). https://doi.org/10.1007/s10561-019-09782-7
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DOI: https://doi.org/10.1007/s10561-019-09782-7