Advertisement

Indian Journal of Clinical Biochemistry

, Volume 34, Issue 1, pp 39–44 | Cite as

Methods for Isolation of High Quality and Quantity of miRNA and Single Cell Suspension for Flow-Cytometry from Breast Cancer Tissue: A Comparative Analysis

  • Shailendra Dwivedi
  • Purvi Purohit
  • Radhieka Misra
  • Puneet Pareek
  • Jeewan Ram Vishnoi
  • Sanjeev Misra
  • Praveen SharmaEmail author
Original Research Article

Abstract

Inadequate methods may cause substantial loss not only in the quantity but also in quality of the product. This study aimed to determine the best method for making single cell suspension for isolation of RNA and flow cytometer analysis from cancer tissue. We compared two methods of tissue disruption used during RNA isolation and flow cytometer analysis. Mechanical tissue disruption method and enzymatic tissue digestion method are commonly used for making single cell suspension before RNA isolation and flow cytometer analysis. 20 resected tissue samples were dissociated into single cells by mechanical and enzymatic methods. Quality and quantity of isolated miRNA was graded by the ratio of 260/280 nm and by running gels. The results revealed that mechanical hand held tissue homogenizer showed better yield than enzymatic (719.12 ± 513.67 vs. 524.87 ± 388.18 ng/µl) and the quality 260/280 nm ratio was significantly better [2.15 ± 0.21 vs. 1.57 ± 0.23; 95% CI (0.402–0.730); p < 0.001] in mechanical method than enzymatic. However, for flow cytometer enzymatic digestion was best. The mechanical method is very suitable for isolating miRNA than enzymatic while enzymatic digestion is most favorable for flow-cytometer analysis as it reduces debris in comparison of mechanical process of shearing.

Keywords

RNA isolation Flow cytometer Mechanical and enzymatic tissue disruption Quality and quantity Debris 

Notes

Acknowledgements

Author Dr. Shailendra Dwivedi is thankful to SERB: Department of Science and Technology, New Delhi for current funding support and fellowship of NPDF: SERB 2015/000322.

Compliance with Ethical Standards

Conflict of interests

The authors declare that they have no competing interests.

References

  1. 1.
    Dwivedi S, Sharma P. Prospects of molecular biotechnology in diagnostics: step towards precision medicine. Indian J Clin Biochem. 2017;32(2):121–3.CrossRefGoogle Scholar
  2. 2.
    Dwivedi S, Purohit P, Misra R, Pareek P, Goel A, Khattri S, Pant KK, Misra Sharma P. Diseases and molecular diagnostics: a step closer to precision medicine. Indian J Clin Biochem. 2017;32(4):374–98.CrossRefGoogle Scholar
  3. 3.
    Dwivedi S, Shukla KK, Gupta G, Sharma P. Non-invasive biomarker in prostate carcinoma: a novel approach. Indian J Clin Biochem. 2013;28(2):107–9.CrossRefGoogle Scholar
  4. 4.
    Dwivedi S, Goel A, Mandhani A, Khattri S, Sharma P, Misra S, Pant KK. Functional genetic variability at promoters of pro-(IL-18) and anti-(IL-10) inflammatory affects their mRNA expression and survival in prostate carcinoma patients: five year follow-up study. Prostate. 2015;75(15):1737–46.CrossRefGoogle Scholar
  5. 5.
    Dwivedi S, Goel A, Khattri S, Mandhani A, Sharma P, Misra S, Pant KK. Genetic variability at promoters of IL-18 pro- and IL-10 anti-inflammatory gene affects susceptibility and their circulating serum levels: an explorative study of prostate cancer patients in North Indian populations. Cytokine. 2015;74(1):117–22.CrossRefGoogle Scholar
  6. 6.
    Dwivedi S, Goel A, Khattri S, Mandhani A, Sharma P, Pant KK. Tobacco exposure by various modes may alter pro-inflammatory (IL-12) and anti-inflammatory (IL-10) levels and affects the survival of prostate carcinoma patients: an explorative study in North Indian population. Biomed Res Int. 2014;2014:158530.CrossRefGoogle Scholar
  7. 7.
    Sharma P, Dwivedi S. Nutrigenomics and nutrigenetics: new insight in disease prevention and cure. Indian J Clin Biochem. 2017;32(4):371–3.CrossRefGoogle Scholar
  8. 8.
    Freshney R. Culture of animal cells: a manual of basic technique. New York: Alan R. Liss, Inc; 1987. p. 117.Google Scholar
  9. 9.
    Bustin SA, Benes V, Nolan T, Pfaffl MW. Quantitative real-time RT-PCR-a. perspective. J Mol Endocrinol. 2005;34:597–601.CrossRefGoogle Scholar
  10. 10.
    Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RTPCR. Nat Protoc. 2006;1:1559–82.CrossRefGoogle Scholar
  11. 11.
    Dolezel J, Greilhuber J, Suda J. Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc. 2007;2:2233–44.CrossRefGoogle Scholar

Copyright information

© Association of Clinical Biochemists of India 2017

Authors and Affiliations

  • Shailendra Dwivedi
    • 1
  • Purvi Purohit
    • 1
  • Radhieka Misra
    • 2
  • Puneet Pareek
    • 3
  • Jeewan Ram Vishnoi
    • 4
  • Sanjeev Misra
    • 4
  • Praveen Sharma
    • 1
    Email author
  1. 1.Department of BiochemistryAll India Institute of Medical SciencesJodhpurIndia
  2. 2.Era’s Lucknow Medical College and HospitalLucknowIndia
  3. 3.Department of Radio-TherapyAll India Institute of Medical SciencesJodhpurIndia
  4. 4.Department of Surgical OncologyAll India Institute of Medical SciencesJodhpurIndia

Personalised recommendations