Heparinase eliminates heparin-associated inhibition of the real-time polymerase chain reaction when assessing gene expression in lung tissue
N. S. Vaсhrushev,
L. A. Shilenko,
Al-Kh. A. Chervaev,
A. A. Karpov,
M. M. Galagudza,
A. A. Kostareva, 
O. V. Kalinina
O. V. Kalinina https://doi.org/10.18705/2311-4495-2024-11-1-55-64
EDN: CFKQDB
Abstract
Background. Histological and genetic methods are often used in the study of pathologies. To prevent contamination of histological preparations with blood, the microvasculature is perfused with a heparin, which leads to its accumulation in the tissue. Heparin is an inhibitor of the polymerase chain reaction (PCR).
Objective. To study the possibility of preventing the influence of unfractionated heparin (UFH), used in perfusion, on the result of gene expression in rat lung samples using RT-PCR by pretreatment RNA with heparinase.
Design and methods. Perfusion of the rats’ vasculature was performed with UFH at concentrations of 50 IU/mL (n = 3) and 500 IU/mL (n = 3), or with a sodium chloride (n = 3) as a control. RNA was isolated from left lung samples and treated with heparinase. The relative expression of five genes (s18, HPRT, Actinβ, GAPDH, Vim) before and after heparinase treatment of isolated RNA preparations was assessed based on the absolute value of the threshold cycle (Ct) obtained by RT-PCR.
Results. UFH increases the threshold cycle level. Treatment of samples with heparinase does not affect the quantity and quality of RNA, but reduce the Ct compared to samples not treated with heparinase.
Conclusion. While planning a study, it is necessary to consider alterations in the results of genetic research that arise due to organ perfusion with a heparin solution. The use of heparinase effectively eliminates heparin-associated RT-PCR inhibition.
Supporting agencies: The research was carried out at the expense of a grant from the Russian Science Foundation No. 23-75-10122, https://rscf.ru/ project/23-75-10122
About the Authors
N. S. Vaсhrushev
Almazov National Medical Research Centre
Russian Federation
Russian Federation
Nikita S. Vaсhrushev, PhD student
Department of Cellular Biology
197341; Akkuratova str., 2; Saint Petersburg
L. A. Shilenko
Almazov National Medical Research Centre
Russian Federation
Russian Federation
Leonid A. Shilenko, first-year resident, Laboratory Assistant
Department of Faculty Therapy with a clinic; Laboratory of Pulmonary Vascular Disease
Saint Petersburg
Al-Kh. A. Chervaev
Almazov National Medical Research Centre
Russian Federation
Russian Federation
Al-Khalim A. Chervaev, first-year resident
Department of Neurosurgery
Saint Petersburg
A. A. Karpov
Almazov National Medical Research Centre; Saint Petersburg Electrotechnical University “LETI”
Russian Federation
Russian Federation
Andrei A. Karpov, PhD, Head of Laboratory, Associate Professor; Leading Researcher
Laboratory of Pulmonary Vascular Disease; Department of Pathology; Department of “Strong Artificial Intelligence Technologies in Physiology and Medicine”
Saint Petersburg
M. M. Galagudza
Almazov National Medical Research Centre
Russian Federation
Russian Federation
Michael M. Galagudza, MD, DSc, Professor, Corresponding Member of the Russian Academy of Science, Director of Institute, Head of the Department
Institute of Experimental Medicine; Department of Pathology
Saint Petersburg
A. A. Kostareva
Almazov National Medical Research Centre; Saint-Petersburg Pasteur Institute
Russian Federation
Russian Federation
Anna A. Kostareva, DSc in Medicine, Director of the Institute
Institute of Molecular Biology and Genetics
Saint Petersburg
O. V. Kalinina
Almazov National Medical Research Centre
Russian Federation
Russian Federation
Olga V. Kalinina, DSc in Biology, Professor
Department of Laboratory Medicine and Genetics
Saint Petersburg
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For citations:
Vaсhrushev N.S., Shilenko L.A., Chervaev A.A., Karpov A.A., Galagudza M.M., Kostareva A.A., Kalinina O.V. Heparinase eliminates heparin-associated inhibition of the real-time polymerase chain reaction when assessing gene expression in lung tissue. Translational Medicine. 2024;11(1):55-64. (In Russ.) https://doi.org/10.18705/2311-4495-2024-11-1-55-64. EDN: CFKQDB
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