Comparison and optimisation of microRNA extraction from the plasma of healthy pregnant women

Abstract

circulating microrna (mirna) biomarkers are implicated in the diagnosis, monitoring and prediction of various disease processes. Before embarking upon biomarker discovery, miRNA extraction techniques must first be optimised in the biofluid and population under study. Using plasma from a healthy pregnant woman, it was attempted to optimise and compare the performance of two commercially available mirna extraction kits; Qiagen (mirneasy Serum/Plasma) and Promega (Maxwell ® rSc mirna from Tissue or Plasma or Serum). Sample mirna content (concentration and percentage) was assessed using agilent Bioanalyzer Small rna chips and reverse transcription-quantitative Pcr (rT-qPcr) using four constitutively expressed mirnas (hsa-mir-222-3p, hsa-let-7i-3p, hsa-mir-148-3p and hsa-mir-30e-5p). Quality control spike-ins monitored rna extraction (uniSp2, 4 and 5) and cdna synthesis (uniSp6, cel-mir-39-3p) efficiency. optimisation approaches included: i) Starting volume of plasma; the addition of ii) Proteinase K; iii) a rna bacteriophage carrier (MS2); and iv) a glycogen carrier. The two kits exhibited equivalence in terms of mirna recovery based on Bioanalyzer and rT-qPcr ΔΔcq results. optimisation attempts for both kits failed to improve upon mirna content compared with standard methodology. comparing the standard methodology, the Qiagen kit was more consistent (smaller variance of Δcq values) compared with the Promega kit. The standard methodology of either kit would be suitable for the investigation of mirna biomarkers in a healthy pregnant population.

Isolation of Cell-Free miRNA from Biological Fluids: Influencing Factors and Methods

“…The similar problem is known for circulating DNA precipitation, for which in unpredictable DNA loss or contamination was shown for some precipitation protocols, especially those which used positively charged compounds [157]. RNA bacteriophage carrier (MS2), yeast RNA, tRNA and glycogen are usually used carriers [137,[158][159][160]. The most common co-precipitator is glycogen; however, the combination of tRNA and glycogen was shown to improve the yield and purity of RNA greatly and to maximize the extraction of miRNA from plasma when using the TRIzol LS [160].…”

miRNA Expression in Ovarian Cancer in Fresh Frozen, Formalin-fixed Paraffin-embedded and Plasma Samples

“…The 2-3 Cp difference exclusion range used by others in the literature and the manufacturer's guidelines (29,44) did not account for variation in dilution. We therefore prepared serial dilutions of the RNA from the two no-tissue controls, providing us with an expected Cp range for the isolation spike-in controls and provided a method for excluding samples with Cp values for UniSP2 outside this range.…”

Towards Personalized Medicine in Myasthenia Gravis: Role of Circulating microRNAs miR-30e-5p, miR-150-5p and miR-21-5p

“…Amplification curves and melting curve analysis were performed to check for correct amplification through the QuantStudio ® Real-Time PCR software (Thermo Fisher Scientific ® , Waltham, MA, USA), samples with a ΔCq of replicas > 1.5 or Ct > 36 were excluded. A ΔCq of replicas > 1.5 was chosen as the cut-off to avoid the exclusion of samples with low miRNA concentration (high Cq), as increasing Cq variance is expected in these cases [ 19 , 20 ]. Majority of replicas (~97.2%) had a ΔCq < 0.5.…”