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

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 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.