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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 PRISMA flow diagram illustrating the search strategy used[98].
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pone.0121948.g003: A PRISMA flow diagram illustrating the search strategy used[98].

Mentions: Of the 220 retrieved abstracts, 130 were excluded because they were reviews, case reports, or non-English language manuscripts. Sixteen publications were unobtainable through our library, leaving 74 manuscripts available for review. A PRISMA flow diagram is shown in Fig 3 and data shown in Table 4 [98]. Although nearly one-third of studies did not mention time to plasma, in the vast majority (nearly 58%), plasma extraction was completed ≤2h after phlebotomy. Most authors used either Trizol, miRNeasy, or mirVana protocols for RNA extraction, and nearly all publications used total miRNA rather than exosomal miRNA. Equal proportions of manuscripts used internal and external references (43% and 42%, respectively). The most commonly utilized internal reference miRNAs were miR-16 (12 publications) and RNU-6 (7 publications). Seventy-one of the 74 reviewed publications did not describe the setting of the cycle threshold bar. All but 2 of the 74 (97%) papers stated at least some aspect of their statistical methods, with many using multiple, different testing methods as shown in Table 4. Among reviewed manuscripts, only 1 (1.4%) listed all 6 assessed criteria [9–84].


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 PRISMA flow diagram illustrating the search strategy used[98].
© Copyright Policy
Related In: Results  -  Collection

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

pone.0121948.g003: A PRISMA flow diagram illustrating the search strategy used[98].
Mentions: Of the 220 retrieved abstracts, 130 were excluded because they were reviews, case reports, or non-English language manuscripts. Sixteen publications were unobtainable through our library, leaving 74 manuscripts available for review. A PRISMA flow diagram is shown in Fig 3 and data shown in Table 4 [98]. Although nearly one-third of studies did not mention time to plasma, in the vast majority (nearly 58%), plasma extraction was completed ≤2h after phlebotomy. Most authors used either Trizol, miRNeasy, or mirVana protocols for RNA extraction, and nearly all publications used total miRNA rather than exosomal miRNA. Equal proportions of manuscripts used internal and external references (43% and 42%, respectively). The most commonly utilized internal reference miRNAs were miR-16 (12 publications) and RNU-6 (7 publications). Seventy-one of the 74 reviewed publications did not describe the setting of the cycle threshold bar. All but 2 of the 74 (97%) papers stated at least some aspect of their statistical methods, with many using multiple, different testing methods as shown in Table 4. Among reviewed manuscripts, only 1 (1.4%) listed all 6 assessed criteria [9–84].

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