MicroRNA profiling in plasma samples using qPCR arrays: Recommendations for correct analysis and interpretation

Abstract

MicroRNA (miRNA) regulate gene expression through posttranscriptional mRNA degradation or suppression of translation. Many (pre)analytical issues remain to be resolved for miRNA screening with TaqMan Low Density Arrays (TLDA) in plasma samples, such as optimal RNA isolation, preamplification and data normalization. We optimized the TLDA protocol using three RNA isolation protocols and preamplification dilutions. By using 100μL elution volume during RNA isolation and adding a preamplification step without dilution, 49% of wells were amplified. Informative target miRNA were defined as having quantification cycle values ≤35 in at least 20% of samples and low technical variability (CV across 2 duplicates of 1 sample <4%). A total of 218 miRNA was considered informative (= 59% of all target miRNA). Different normalization strategies were compared: exogenous Ath-miR-159a, endogenous RNA U6, and three mathematical normalization techniques: geNorm (Qbase, QB) and NormFinder (NF) normalization algorithms, and global mean calculation. To select the best normalization method, technical variability, biological variability, stability, and the extent to which the normalization method reduces data dispersion were calculated. The geNorm normalization algorithm reduced data dispersion to the greatest extent, while endogenous RNA U6 performed worst. In conclusion, for miRNA profiling in plasma samples using TLDA cards we recommend: 1. Implementing a preamplification step in the TLDA protocol without diluting the final preamplification product 2. A stepwise approach to exclude non-informative miRNA based on quality control parameters 3. Against using snoRNA U6 as normalization method for relative quantification 4. Using the geNorm algorithm as normalization method for relative quantification.

Enhanced GIP Secretion in Obesity Is Associated with Biochemical Alteration and miRNA Contribution to the Development of Liver Steatosis

“…Another limitation of our study is the small number of samples available for miRNA profiling. Nevertheless, we made an attempt to carry out miRNA analysis in subjects representative for both high GIP and low GIP groups, in accordance with most current guidelines [36]. For this reason, we suggest that miRNAs differentially expressed, according to circulating GIP, in our study, should be validated on a larger group of subjects.…”

“…The arrays were run on the 7900HT Fast Real-Time PCR system (ThermoFisher, Waltham, MA, USA). According to recommendations [36], a miRNA was considered non-informative if C T values were >35 in >80% of samples. Relative miRNA levels were expressed as fold of change (fold of change (RQ) = geometric mean 2 (−∆Ct high GIP) / geometric mean 2 (−∆Ct lowGIP) ), where U6 snRNA was used as endogenous control.…”

MicroRNA Isolation from Plasma for Real‐Time qPCR Array

“…1). In our optimizing experiments for microRNA arrays, it appeared that preamplification of the cDNA was an essential step in the procedure to obtain a larger quantity of amplified wells (Gevaert et al, 2018). The protocol is based on the Megaplex Pools With Preamplification protocol for miRNA Expression Analysis (Applied Biosystems) and was modified only slightly.…”

“…In this article, particular attention is paid to the processing and separation of plasma samples, total RNA extraction from plasma samples, and further analysis using reverse transcription, preamplification, and real-time qPCR for TaqMan Low Density Arrays (TLDA). This article is based on previously published results regarding the comparison of different strategies for RNA isolation from plasma, preamplification and dilution of the preamplification product, as well as quantification using TaqMan Low Density Arrays (Gevaert et al, 2018). The recommendations of Gevaert et al on miRNA isolation and quantification are the following: (1) use Arabidopsis thaliana (Ath) miR-159a as spike-in control, (2) use a 100-µl elution volume during RNA isolation, and (3) add a preamplification step without dilution of the preamplification product.…”

MicroRNAs in AKI and Kidney Transplantation

“…Several caveats apply when interpreting the study results. First, the considerable heterogeneity in the techniques used (quantitative RT-PCR, microarrays, next generation sequencing) (10) and patient groups studied makes intergroup comparisons and independent validation difficult. Second, several of these studies investigated microRNA target prediction and mRNA interactions through biostatistical modeling.…”