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. 2019 Jan;34(1):39-44.
doi: 10.1007/s12291-017-0719-5. Epub 2017 Nov 29.

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  1 , Purvi Purohit  1 , Radhieka Misra  2 , Puneet Pareek  3 , Jeewan Ram Vishnoi  4 , Sanjeev Misra  4 , Praveen Sharma  1
Affiliations

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 et al. Indian J Clin Biochem. 2019 Jan.

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

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Conflict of interest statement

Compliance with Ethical StandardsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Agarose gel showing RNA bands, tissues were disrupted by mechanical method (handheld homogenizer) A and C samples were 2000 mg, others were of 1000 mg weight
Fig. 2
Fig. 2
Agarose gel showing RNA bands, tissues were disrupted by enzymatic method, where A, E, H were digested by trypsin and remaining by collagenase, A and C samples were 2000 mg, others were of 1000 mg weight
Fig. 3
Fig. 3
Quality assessment of isolated RNA after two different method of tissue disruption
Fig. 4
Fig. 4
Dot plot diagram of Flow-cytometer showing the difference in debris, first diagram showed mechanical method having more debris than enzymatic method (second diagram on right) of tissue disruption
See this image and copyright information in PMC

References

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