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Assay reproducibility in clinical studies of plasma miRNA.

Rice J, Roberts H, Burton J, Pan J, States V, Rai SN, Galandiuk S - PLoS ONE (2015)

Bottom Line: We compared these data with a proposed standard methodologic technique.There was no significant intra-operator variability.There was significant inter-operator variation using Trizol LS extraction, while this was negligible using modified miRNeasy.

View Article: PubMed Central - PubMed

Affiliation: Price 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.

No MeSH data available.


Related in: MedlinePlus

The phases and components of a PCR curves: (1) baseline, (2) exponential phase, (3) linear phase, (4) plateau phase, and (5) cycle threshold.
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pone.0121948.g002: The phases and components of a PCR curves: (1) baseline, (2) exponential phase, (3) linear phase, (4) plateau phase, and (5) cycle threshold.

Mentions: In order to compare cycle threshold (Ct) values across plates, both fixed and variable thresholds were utilized. Since different miRNAs on the same plate may have different linear phases, a fixed threshold may not intersect across the linear phase. For this reason, the variable threshold is the default setting. Cycle threshold is illustrated in Fig 2, which shows the phases of the PCR curves: (1) the baseline, (2) the exponential phase, (3) the linear phase, and 4) the plateau phase. The RQ manager from Applied Biosystems may use a different threshold (variable) within the same plate if one selects the option “automatic Ct”. Different thresholds will be chosen for different miRNAs according to the linear phases. However, these Ct values cannot be compared directly between different plates. The threshold needs to be considered in the analysis and adjustment of the Ct values is needed. In addition to this variable threshold setting, we examined the effect of fixed threshold settings of 0.01, 0.03, 0.05, or 0.5. Ct values, threshold, and 40 cycles fluorescence intensities are miRNA-rated from RQ manager 1.2, Applied Biosystems.


Assay reproducibility in clinical studies of plasma miRNA.

Rice J, Roberts H, Burton J, Pan J, States V, Rai SN, Galandiuk S - PLoS ONE (2015)

The phases and components of a PCR curves: (1) baseline, (2) exponential phase, (3) linear phase, (4) plateau phase, and (5) cycle threshold.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390277&req=5

pone.0121948.g002: The phases and components of a PCR curves: (1) baseline, (2) exponential phase, (3) linear phase, (4) plateau phase, and (5) cycle threshold.
Mentions: In order to compare cycle threshold (Ct) values across plates, both fixed and variable thresholds were utilized. Since different miRNAs on the same plate may have different linear phases, a fixed threshold may not intersect across the linear phase. For this reason, the variable threshold is the default setting. Cycle threshold is illustrated in Fig 2, which shows the phases of the PCR curves: (1) the baseline, (2) the exponential phase, (3) the linear phase, and 4) the plateau phase. The RQ manager from Applied Biosystems may use a different threshold (variable) within the same plate if one selects the option “automatic Ct”. Different thresholds will be chosen for different miRNAs according to the linear phases. However, these Ct values cannot be compared directly between different plates. The threshold needs to be considered in the analysis and adjustment of the Ct values is needed. In addition to this variable threshold setting, we examined the effect of fixed threshold settings of 0.01, 0.03, 0.05, or 0.5. Ct values, threshold, and 40 cycles fluorescence intensities are miRNA-rated from RQ manager 1.2, Applied Biosystems.

Bottom Line: We compared these data with a proposed standard methodologic technique.There was no significant intra-operator variability.There was significant inter-operator variation using Trizol LS extraction, while this was negligible using modified miRNeasy.

View Article: PubMed Central - PubMed

Affiliation: Price 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.

No MeSH data available.


Related in: MedlinePlus