Open AccessDevelopment of a method for determining the levels of normalized expression of structural and functional genes in patients with knee joint arthropathy
Author(s) -
С. А. Костюк,
O. S. Poluyan,
Т. В. Руденкова,
A. N. Benko,
М. А. Герасименко
Publication year - 2020
Publication title -
problemy zdorovʹâ i èkologii
Language(s) - English
Resource type - Journals
eISSN - 2708-6011
pISSN - 2220-0967
DOI - 10.51523/2708-6011.2020-17-3-22
Subject(s) - gene , reverse transcriptase , aggrecanase , multiplex , computational biology , microbiology and biotechnology , oligonucleotide , reverse transcription polymerase chain reaction , biology , gene expression , bioinformatics , genetics , osteoarthritis , medicine , polymerase chain reaction , pathology , articular cartilage , alternative medicine
Objective : to develop a method of molecular genetic analysis for determining the normalized expression levels of COL2A1, COL6A1, MMP-2, and MMP-9 genes in the biological material of patients with gonarthrosis. Material and methods . The biopsy samples of knee joint cartilage of 10 patients with gonarthrosis were used as a biological material for the study. Nucleic acids were isolated from the samples of the biological material using TRIZol Reagent. Reverse transcription was performed using the SuperScript III reverse transcriptase kit, dNTP and Ribonuclease inhibitor (Invitrogen, USA). Results . The usage of VectorNTI software has made it possible to select pairs of primers (forward and reverse) and TaqMan probes for COL2A1, COL6A1, MMP-2 , and MMP-9 genes, and to study the possibility of the use of the selected pairs of primers and probes for the purpose of identification of each of the studied genes. In-house test systems in the multiplex format have been designed for reference and target genes. Conclusion . High specificity (100 %) of the selected oligonucleotide primer sets was confirmed using the online application NCBI/Blast. The developed molecular genetic method can be applied to determine the normalized expression levels of COL2A1, COL6A1, MMP-2 , and MMP-9 genes in knee joint arthropathy