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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

A) Receiver operator characteristic (ROC) curve for miR-523,miR-218,miR-142-3p,miR 27a,miR-21. Colorectal cancer (n = 20) vs. Breast cancer +Pancreatic cancer + Lung cancer (n = 30 [10 each group]). B) ROC Curve for miR-523, miR-218, miR-142-3p,miR-27a,miR-376c,miR-374. Colorectal cancer (n = 20) + Colorectal adenoma (n = 10) vs. Breast cancer +Pancreatic cancer + Lung cancer (n = 30 [10 each group]).
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pone.0121948.g001: A) Receiver operator characteristic (ROC) curve for miR-523,miR-218,miR-142-3p,miR 27a,miR-21. Colorectal cancer (n = 20) vs. Breast cancer +Pancreatic cancer + Lung cancer (n = 30 [10 each group]). B) ROC Curve for miR-523, miR-218, miR-142-3p,miR-27a,miR-376c,miR-374. Colorectal cancer (n = 20) + Colorectal adenoma (n = 10) vs. Breast cancer +Pancreatic cancer + Lung cancer (n = 30 [10 each group]).

Mentions: With respect to choice of number of miRNA in our panel, it was our expectation that no more than 10% of miRNA would be differentially expressed between cases and controls after adjusting the p values for multiple comparisons. Of these, in turn, one would not expect more that 0.5 to 3% of miRNA to be able to accurately identify cases and controls. Ten miRNA and one reference (housekeeping gene) miRNA were, therefore, chosen (approximately 3%). Statistical analysis using ANOVA identified 11 significantly dysregulated miRNAs specific for colorectal neoplasia (Table 2). Multiple test control was based on controlling the false discovery rate (FDR) at 10%. A logistic regression model was established using the top up-regulated miRNAs and used for predicting the adenoma and control groups for the validation data. The sensitivity and specificity for this prediction were calculated. The receiver operator characteristic (ROC) curves with AUC values were microRNA-rated using current versions of SAS [85] and R [86–88] (Fig 1a & 1b). These data are not the focus of this report; however, these selected miRNA were utilized for the present study to evaluate the effect of time to plasma extraction, the effect of multiple samples drawn from an individual over time, the method of RNA extraction, cycle threshold bar setting, and intra- as well as inter-operator variability.


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)

A) Receiver operator characteristic (ROC) curve for miR-523,miR-218,miR-142-3p,miR 27a,miR-21. Colorectal cancer (n = 20) vs. Breast cancer +Pancreatic cancer + Lung cancer (n = 30 [10 each group]). B) ROC Curve for miR-523, miR-218, miR-142-3p,miR-27a,miR-376c,miR-374. Colorectal cancer (n = 20) + Colorectal adenoma (n = 10) vs. Breast cancer +Pancreatic cancer + Lung cancer (n = 30 [10 each group]).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0121948.g001: A) Receiver operator characteristic (ROC) curve for miR-523,miR-218,miR-142-3p,miR 27a,miR-21. Colorectal cancer (n = 20) vs. Breast cancer +Pancreatic cancer + Lung cancer (n = 30 [10 each group]). B) ROC Curve for miR-523, miR-218, miR-142-3p,miR-27a,miR-376c,miR-374. Colorectal cancer (n = 20) + Colorectal adenoma (n = 10) vs. Breast cancer +Pancreatic cancer + Lung cancer (n = 30 [10 each group]).
Mentions: With respect to choice of number of miRNA in our panel, it was our expectation that no more than 10% of miRNA would be differentially expressed between cases and controls after adjusting the p values for multiple comparisons. Of these, in turn, one would not expect more that 0.5 to 3% of miRNA to be able to accurately identify cases and controls. Ten miRNA and one reference (housekeeping gene) miRNA were, therefore, chosen (approximately 3%). Statistical analysis using ANOVA identified 11 significantly dysregulated miRNAs specific for colorectal neoplasia (Table 2). Multiple test control was based on controlling the false discovery rate (FDR) at 10%. A logistic regression model was established using the top up-regulated miRNAs and used for predicting the adenoma and control groups for the validation data. The sensitivity and specificity for this prediction were calculated. The receiver operator characteristic (ROC) curves with AUC values were microRNA-rated using current versions of SAS [85] and R [86–88] (Fig 1a & 1b). These data are not the focus of this report; however, these selected miRNA were utilized for the present study to evaluate the effect of time to plasma extraction, the effect of multiple samples drawn from an individual over time, the method of RNA extraction, cycle threshold bar setting, and intra- as well as inter-operator variability.

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