Include variations in plasma samples, collection method, anticoagulant used, inhibitors in plasma
FRIDAY, Dec. 9 (HealthDay News) -- Accurate detection and quantitation of circulating microRNAs (miRNAs) is affected by inherent differences in plasma samples, methods used for their collection and analysis, and the presence of specific inhibitors in plasma, according to a study published online Dec. 9 in the Journal of Molecular Diagnostics.
Dong-Ja Kim, from the Chicago Medical School, and colleagues standardized and optimized miRNA detection for biomarker studies by quantifying two miRNAs: miR-16 (tumor suppressor) and miR-223 (implicated in pregnancy, other conditions, and malignant disease). Blood samples were collected and processed, and RNA was isolated. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative-PCR were used to measure miRNA abundance.
The investigators found that the reproducibility of miRNA quantitation was affected by the collection method and anticoagulants used (best results obtained by collecting blood into tubes with sodium fluoride/potassium oxalate [NaF/KOx]), irrespective of the abundance of the miRNA in blood plasma. The anticoagulant used affected miRNA stability and sensitivity of miRNA detection; NaF/KOx was thought to enable enhanced sensitivity in quantitation. Enhancement of small RNAs effectively removed blood-borne reverse transcriptases and/or PCR inhibitors, thereby improving the accuracy of miRNA quantitation. Other factors which impacted the accuracy of quantitation included careful titration of starting materials, heparinase treatment which increases miRNA detection in heparinized plasma, and differences in plasma composition among individual donors.
"The implications of the work are that without consideration of the variables we have identified, miRNA quantitation of human samples may not be reliable for the purpose of biomarker development," the authors write.