Optimization of a library preparation protocol for sequencing circulating plasma and serum microRNAs
A. V. Kiseleva,
E. A. Sotnikova,
A. A. Zharikova,
V. A. Kutsenko,
A. L. Borisova,
S. A. Shalnova,
A. I. Ershova,
A. N. Meshkov,
O. M. Drapkina https://doi.org/10.15829/1728-8800-2025-4557
EDN: DVGAAK
Abstract
Aim. To optimize a library preparation protocol for sequencing small non-coding ribonucleic acids (microRNAs) based on the commercial QIAseq miRNA UDI Library Kit to improve the quality of the obtained data.
Material and methods. Plasma and serum samples from four study participants were collected from the biobank collection of the National Medical Research Center for Therapeutic and Preventive Medicine. Ribonucleic acid (RNA) was isolated for each sample in parallel, using 200 and 300 µl aliquots. Two sequencing libraries were prepared from each RNA sample using the QIAseq miRNA UDI Library Kit by two manufacturer's protocol versions as follows: one for 1 ng of RNA with a reduced number of amplification cycles and one for 10 ng of RNA. Sequencing was performed on a NextSeq 550.
Results. When comparing groups of samples prepared using different protocol versions, there was a significant difference in the tags per million reads (TPM) per sample for human (ENCODE v47) and microRNA genes (p<0,001).
Conclusion. We showed that when using plasma and serum biosamples, the main parameter influencing higher microRNA sequencing rates is a reduction in the number of polymerase chain reaction cycles during library preparation.
Supporting agencies: State assignment "Circulating MicroRNAs in Plasma as Diagnostic and Prognostic Biomarkers of CAD"
About the Authors
A. V. Kiseleva
National Medical Research Center for Therapy and Preventive Medicine
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990
E. A. Sotnikova
National Medical Research Center for Therapy and Preventive Medicine
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990
A. A. Zharikova
National Medical Research Center for Therapy and Preventive Medicine; Lomonosov Moscow State University
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990; Leninskiye Gory, 1, Moscow, 119234
V. A. Kutsenko
National Medical Research Center for Therapy and Preventive Medicine
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990
A. L. Borisova
National Medical Research Center for Therapy and Preventive Medicine
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990
S. A. Shalnova
National Medical Research Center for Therapy and Preventive Medicine
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990
A. I. Ershova
National Medical Research Center for Therapy and Preventive Medicine
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990
A. N. Meshkov
National Medical Research Center for Therapy and Preventive Medicine
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990
O. M. Drapkina
National Medical Research Center for Therapy and Preventive Medicine
Russian Federation
Russian Federation
Petroverigsky Lane, 10, bld. 3, Moscow, 101990
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Supplementary files
What is already known about the subject?
- Next-generation sequencing provides high sensitivity and specificity, as well as the ability to quantify and detect new small non-coding ribonucleic acids (microRNAs).
- Library preparation for microRNA sequencing can significantly impact the results obtained, including the coverage and profile of detected microRNAs.
What might this study add?
- When using low miRNA content biosamples in research, optimization of the sequencing library preparation protocol can significantly improve the results obtained.
Review
For citations:
Kiseleva A.V., Sotnikova E.A., Zharikova A.A., Kutsenko V.A., Borisova A.L., Shalnova S.A., Ershova A.I., Meshkov A.N., Drapkina O.M. Optimization of a library preparation protocol for sequencing circulating plasma and serum microRNAs. Cardiovascular Therapy and Prevention. 2025;24(11):4557. (In Russ.) https://doi.org/10.15829/1728-8800-2025-4557. EDN: DVGAAK
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