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Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR
Published on: February 12, 2018
Assay reproducibility in clinical studies of plasma miRNA
Jonathan Rice1, Henry Roberts1, James Burton1
1Price Institute of Surgical Research, Hiram C. Polk Jr., M.D. Department of Surgery, University of Louisville School of Medicine, Louisville, KY, United States of America.
Abstract:
There are increasing reports of plasma miRNAs as biomarkers of human disease but few standards in methodologic reporting, leading to inconsistent data. We systematically reviewed plasma miRNA studies published between July 2013-June 2014 to assess methodology. Six parameters were investigated: time to plasma extraction, methods of RNA extraction, type of miRNA, quantification, cycle threshold (Ct) setting, and methods of statistical analysis. We compared these data with a proposed standard methodologic technique. Beginning with initial screening for 380 miRNAs using microfluidic array technology and validation in an additional cohort of patients, we compared 11 miRNAs that exhibited differential expression between 16 patients with benign colorectal neoplasms (advanced adenomas) and 16 patients without any neoplasm (controls). Plasma was isolated immediately, 12, 24, 48, or 72 h following phlebotomy. miRNA was extracted using two different techniques (Trizol LS with pre-amplification or modified miRNeasy). We performed Taqman-based RT-PCR assays for the 11 miRNAs with subsequent analyses using a variable Ct setting or a fixed Ct set at 0.01, 0.03, 0.05, or 0.5. Assays were performed in duplicate by two different operators. RNU6 was the internal reference. Systematic review yielded 74 manuscripts meeting inclusion criteria. One manuscript (1.4%) documented all 6 methodological parameters, while < 5% of studies listed Ct setting. In our proposed standard technique, plasma extraction ≤12 h provided consistent ΔCt. miRNeasy extraction yielded higher miRNA concentrations and fewer non-expressed miRNAs compared to Trizol LS (1/704 miRNAs [0.14%] vs 109/704 miRNAs [15%], not expressed, respectively). A fixed Ct bar setting of 0.03 yielded the most reproducible data, provided that <10% miRNA were non-expressed. There was no significant intra-operator variability. There was significant inter-operator variation using Trizol LS extraction, while this was negligible using modified miRNeasy. For standardized reporting, we recommend plasma extraction ≤ 12 h, using modified miRNeasy extraction and utilizing a 0.03 Ct.
Insights
Standardizing plasma microRNA (miRNA) analysis is crucial for reliable disease biomarker discovery. This study recommends specific methods for plasma extraction, RNA extraction, and cycle threshold settings to improve data consistency in miRNA research.
Area of Science:
- Biochemistry
- Molecular Biology
- Biomarker Discovery
Background:
- Plasma microRNAs (miRNAs) show promise as biomarkers for human diseases.
- Inconsistent reporting of methodologies in plasma miRNA studies leads to unreliable data.
- Lack of standardized protocols hinders the clinical translation of miRNA biomarkers.
Purpose of the Study:
- To systematically review methodologies in plasma miRNA studies.
- To identify critical parameters affecting data reproducibility.
- To propose a standardized method for plasma miRNA analysis.
Main Methods:
- Systematic review of 74 plasma miRNA studies (July 2013-June 2014).
- Investigation of six methodological parameters: plasma extraction time, RNA extraction method, miRNA type, quantification, cycle threshold (Ct) setting, and statistical analysis.
- Comparison of proposed standard techniques with existing study methods.
Main Results:
- Only 1.4% of reviewed studies documented all six methodological parameters.
- Plasma extraction within 12 hours ensured consistent ΔCt values.
- Modified miRNeasy extraction yielded higher miRNA concentrations and fewer non-expressed miRNAs compared to Trizol LS.
- A fixed Ct bar setting of 0.03 provided the most reproducible data.
- Negligible inter-operator variability was observed with modified miRNeasy extraction.
Conclusions:
- Standardized reporting of plasma miRNA studies is urgently needed.
- Recommended standardized methods include plasma extraction ≤12 hours, modified miRNeasy RNA extraction, and a 0.03 Ct setting.
- Adoption of these standards will enhance the reliability and reproducibility of plasma miRNA biomarker research.
