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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

Heat map showing expression of 11 miRNA in plasma of 16 patients with colorectal adenoma prior to treatment and in plasma of 16 controls.Color gradation refers to delta Ct values with RNU6 as reference. Negative values represent over-expression of the target miRNA in comparison to the reference miRNA (RNU6).
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pone.0121948.g004: Heat map showing expression of 11 miRNA in plasma of 16 patients with colorectal adenoma prior to treatment and in plasma of 16 controls.Color gradation refers to delta Ct values with RNU6 as reference. Negative values represent over-expression of the target miRNA in comparison to the reference miRNA (RNU6).

Mentions: The Qiagen miRNeasy extraction without pre-amplification resulted in a higher yield of RNA than Trizol LS extraction; therefore, pre-amplification was not necessary and could be omitted as a source of possible variation (Table 11). The average ΔCT was lower with mirNeasy, with fewer missing values than with Trizol purification and pre-amplification. Qiagen miRNeasy extraction without pre-amplification resulted in only 1of 704 (0.14%) miRNAs not expressing; however, utilizing the Trizol LS extraction with pre-amplification, 109 of 704 (15%) of miRNAs investigated were not expressed (Table 9). A heat map showing miRNA expression for patient groups is shown in Fig 4.


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)

Heat map showing expression of 11 miRNA in plasma of 16 patients with colorectal adenoma prior to treatment and in plasma of 16 controls.Color gradation refers to delta Ct values with RNU6 as reference. Negative values represent over-expression of the target miRNA in comparison to the reference miRNA (RNU6).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0121948.g004: Heat map showing expression of 11 miRNA in plasma of 16 patients with colorectal adenoma prior to treatment and in plasma of 16 controls.Color gradation refers to delta Ct values with RNU6 as reference. Negative values represent over-expression of the target miRNA in comparison to the reference miRNA (RNU6).
Mentions: The Qiagen miRNeasy extraction without pre-amplification resulted in a higher yield of RNA than Trizol LS extraction; therefore, pre-amplification was not necessary and could be omitted as a source of possible variation (Table 11). The average ΔCT was lower with mirNeasy, with fewer missing values than with Trizol purification and pre-amplification. Qiagen miRNeasy extraction without pre-amplification resulted in only 1of 704 (0.14%) miRNAs not expressing; however, utilizing the Trizol LS extraction with pre-amplification, 109 of 704 (15%) of miRNAs investigated were not expressed (Table 9). A heat map showing miRNA expression for patient groups is shown in Fig 4.

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