ISSN Print 2500–1094
ISSN Online 2542–1204
BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)
Copyright: © 2026 by the authors. Licensee: Pirogov University.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (CC BY).
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (CC BY).
ORIGINAL RESEARCH
A reliable and reproducible multiplex RT-qPCR assay for mTOR gene expression analysis
About authors
1 Ural State Medical University, Yekaterinburg, Russia
2 Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg, Russia
3 State Autonomous Health Institution of the Sverdlovsk Region “Center for Specialized Types of Medical Care ‘Institute of Medical Cellular Technologies’”, Yekaterinburg, Russia
4 Medical Center “Garmoniya”, Yekaterinburg, Russia
Correspondence should be addressed: Daniil O. Kornilov
Klyuchevskaya, 17, Yekaterinburg, 620109, Russia; moc.liamg@linrokvolinad
About paper
Acknowledgments: The authors express their gratitude to the staff of the Laboratory of Enteral Viral Infections, Federal Budgetary Institution of Science “Federal Scientific Research Institute of Viral Infections ‘Virome’” of Rospotrebnadzor, for their assistance in organizing the work.
Author contribution: Kornilov DO — conceptualization, methodology, writing — original draft; Simarzina VM — investigation, methodology, visualization; Bekhter AA — investigation, visualization; Maslakov GP — investigation, software, validation; Nechaeva DM — methodology, writing — review and editing; Kariakina AE — methodology, writing — review and editing; Fadeev FA — resources, writing — review and editing; Voroshilina ES — supervision, project administration; Zornikov DL — supervision, visualization, formal analysis, writing — review and editing.
Compliance with ethical standards: The study was approved by the Local Ethical Committee of the Ural State Medical University, Yekaterinburg, Russia (Protocol No. 6 dated October 18, 2024). Informed consent was not applicable, as commercial cell lines were used.
Received: 2026-03-11
Accepted: 2026-03-25
Published online: 2026-03-25
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Fig. 1. Experimental design for multiplex RT‑qPCR assay validation. The scheme illustrates the two independent dilution series used to assess assay performance. The pre‑analytical series (yellow) involved total RNA isolation from SCP‑1 cells at five different starting concentrations. The analytical series (blue) was created by twofold serial dilution of the RNA isolated from the highest cell concentration. Each resulting RNA sample (n = 9) was analyzed in triplicate, totaling 27 individual reactions for the evaluation of PCR efficiency, analytical sensitivity, and technical reproducibility
Fig. 2. Results of amplification product specificity analysis by agarose gel electrophoresis. A. Separate RT-qPCR technical repeats for each target gene. B. Multiplex RT-qPCR technical repeats amplifying all three targets simultaneously. Lanes for each gene show a single amplicon corresponding to the expected size, confirming high assay specificity
Fig. 3. Amplification efficiency of the target gene mTOR and reference genes RPLP0 and TBP. Amplification efficiency analysis for mTOR, RPLP0, and TBP genes showing linear regression plots of Cp values versus log2 of the Cell Equivalent. Experimental amplification efficiencies were 81% for mTOR (regression equation: y = 39 – 0.81x), 79% for RPLP0 (y = 33 – 0.79x), and 73% for TBP (y = 41 – 0.73x). The dashed red line represents theoretical 100% amplification efficiency (slope = –1), when Cp value decreases by one for each twofold increase of cell concentration, while solid black lines show actual regression lines, and black dots indicate individual data points from multiplex RT‑qPCR technical repeats
Fig. 4. Analysis of mTOR expression relative to reference genes RPLP0 and TBP. The expression of mTOR was consistently lower than RPLP0 and higher than TBP in the SCP-1 cell line
Fig. 5. Assessment of analytical reproducibility using different normalization strategies. The percentage deviation of FC values for each sample from the overall median. The reproducibility of results when mTOR expression normalized to RPLP0, TBP, and their geometric mean (RPLP0 and TBP). The reduced variability (narrower interquartile range) achieved by using two reference genes simultaneously
Table. Oligonucleotide characteristics for the multiplex RT-qPCR assay
Note: F — forward; R — reverse. Gene symbols refer to mammalian target of rapamycin (mTOR), ribosomal protein lateral stalk subunit P0 (RPLP0), and TATA‑box binding protein (TBP). All primers and probes were designed to meet standard criteria for multiplex real‑time PCR, including amplicon size (<150 bp), matched melting temperatures within each set, optimal GC‑content, and distinct, non‑overlapping fluorescent detection channels
