Evaluating the influence of different anticoagulant blood collection tubes on the efficiency of circulating RNA extraction and Real-Time PCR quantification

Abstract Background and aims Rigorous pre-analytical standardization is a prerequisite for reliable use of circulating microRNAs and RNA as clinical biomarkers. This study evaluates the influence of four common anticoagulants (sodium citrate, Dipotassium Ethylenediaminetetraacetic Acid (K2-EDTA), Lithium heparin, and Acid Citrate Dextrose (ACD)) on isolation and quantification of miRNA and RNA from human plasma. Materials and methods Fifteen healthy volunteers were recruited from the research staff and donated blood to study the effects of different anticoagulants on molecular tests. After Trizol extraction, we assessed RNA quantification and purity via spectrophotometry and amplification efficiency via Real-Time PCR for miR-133a, cel-miR-39 (exogenous spike-in), and U6snRNA (endogenous control). Results Our results confirmed that while K2-EDTA, sodium citrate and ACD provide comparable enzymatic environments for Real-time PCR, lithium heparin strongly inhibited reverse transcriptase and DNA polymerase activity, preventing successful amplification. Furthermore, specific differences in spectrophotometric purity ratios (A 260 /A 280 ) were identified, suggesting that the chemical nature of the anticoagulant subtly influences the UV absorbance profile of the extract and its purity. Conclusions For researchers focusing on circulating RNA and miRNA, sodium citrate, ACD and K2-EDTA should be regarded as suitable anticoagulants, with sodium citrate and ACD offering slightly better spectrophotometric purity profiles. Lithium heparin is strongly discouraged for studies involving enzymatic amplification (PCR), as the Trizol extraction method is insufficient to remove the inhibitory heparin molecules.