Table 3 A summary of the authors’ recommendations for standardized methods in circulating miRNA profiling

Pre-analytical

Sampling

Freeze samples at − 80 °C (or at least − 20 °C) as soon as possible within a standardized time range for all samples. We suggest within 1 h

Pre-analytical

Centrifugation

Centrifuge samples to eliminate circulating cells or debris under standardized settings (speed, temperature), using the same centrifuge if possible. We suggest 2000g, at 20–25 °C for 15 min

Pre-analytical

Hemolysis detection

Inspect presence of hemolysis by spectrophotometric absorbance at 414 nm, or by monitoring qPCR miR ratio between miR23a and miR 451a

Pre-analytical

Carrier

Use a carrier during RNA extraction, for fluids expected to contain low levels of miRNAs (such CSF, serum, urine). We suggest MS2 phage RNA carrier

Pre-analytical

cDNA synthesis

Perform 2–3 replicates for each RNA stock to detect possible inhibitors carried over from RNA isolation

Pre-analytical

Spike-in

Use an exogenous miRNAs (e.g. Cel-miR-39a) to add prior to RNA isolation or cDNA synthesis to assess technical performance

Pre-analytical

Primers

Re-design primers with PCR efficiencies outside the range of 80–110%

Analytical

Normalization

For normalization, several miRNAs should be tested for stability between controls and disease samples using suitable software algorithms, for example GeNorm and/or NormFinder. We recommend using two or more miRNAs for normalization if possible

Analytical

Statistics

Normal distributed data: use parametric test (e.g. t-test, ANOVA)

Data not normal distributed: use non-parametric test (e.g. Mann–Whitney test)

Apply more complex model (seek professional statistical assistance) if the model has several confounding variables (gender, age, etc.)

Correct P values for multiple testing