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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

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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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Correspondence to Poonam B. Gera or Sanjay Gupta.

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All authors declare that there is no conflict of interest.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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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)

Supplementary Fig. 2: RIN data for ten human tissue samples (TIFF 5448 kb)

Supplementary Fig. 3: RIN data for ten human tissue samples (TIFF 5505 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

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