Assay reproducibility in clinical studies of plasma miRNA

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.

Fig 1

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]).

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

Fig 3. A PRISMA flow diagram illustrating the search strategy used[98].

Fig 4. 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).