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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The PI3K/AKT/mTOR signaling pathway is a key regulator of cell growth, and its dysregulation is involved in oncogenesis. Existing methods for assessing mTOR activity have design flaws. The aim of this work was to develop and validate a novel multiplex RT-qPCR assay for relative quantification of mTOR gene expression normalized to RPLP0 and TBP. Primers and probes were designed in silico. Validation was performed using the human SCP-1 cell line. Specificity was assessed in 10 separate and 10 multiplex runs. Analytical sensitivity and efficiency were determined from 27 technical replicates using a protocol without an elongation step. Specificity of amplification was assessed by agarose gel electrophoresis, and quantitative analysis was performed in real-time PCR using FAM (mTOR), HEX (RPLP0), and ROX (TBP) fluorescence channels. The assay showed 100% specificity. Stable detection was achieved at 125,000 cells/mL. Amplification efficiencies were 73–81%. The variation of mTOR expression normalized to RPLP0 ranged from –21.5% to 26.4%, and normalized to TBP from –14.3% to 19.2%. Normalization to the geometric mean of both reference genes provided the best reproducibility, with an interquartile range from –9% to 23.4%. The developed assay demonstrates high specificity, sensitivity, and reproducibility, making it a reliable tool for subsequent clinical research.
Keywords: molecular diagnostics, gene expression profiling, multiplex polymerase chain reaction, reproducibility of results, reverse transcriptase polymerase chain reaction, TOR serine-threonine kinases