Circumventing qPCR inhibition to amplify miRNAs in plasma

Abstract

BackgroundCirculating microRNAs (c-miRNAs) have be identified in saliva, urine and blood, which has led to increasing interest in their development as biomarkers for diverse diseases including cancers. One of the key advantages of c-miRNAs over other biomarkers is the ability to be amplified and quantified by quantitative PCR (qPCR). However, at phlebotomy when whole blood is dispensed into heparinized tubes, residual levels of the anti-coagulant lithium heparin may remain in the plasma and hence with RNA isolated from the plasma. This can confound the detection of c-miRNAs by qPCR because it inhibits reverse transcriptase (RT). Here we present a procedure, modified from earlier techniques, to detect c-miRNAs in plasma that improves sensitivity and streamlines performance.FindingsTreatment of total RNA isolated from human blood plasma with Bacteroides heparinase I during reverse transcription at 37°C for one hour improved sensitivity and performance of the qPCR. This is in comparison to no treatment or treatment of the RNA prior to RT, which is the current suggested method and exposes plasma to Flavobacterium heparinum heparinase I for up to 2 hours before RT. This modest alteration improved qPCR performance and resulted in lowered threshold cycles (Ct) for detection of the target sequence, candidate c-miRNA biomarkers, and controls. It also reduced the expense and number of processing steps, shortening the duration of the assay and minimizing exposure of RNA to elevated temperatures.ConclusionIncorporating Bacteroides heparinase I treatment into conventional RT protocols targeting c-miRNA in plasma can be expected to expedite the discovery of biomarkers.

Maternal pre-pregnancy body mass index and circulating microRNAs in pregnancy

“…RNA quality was assessed using threshold cycle (Ct) measurement of two endogenous miRNAs (miR-150 and miR-451) and a synthetic miRNA sequence, cel-miR-257, in randomly selected samples from each batch. Samples with Ct<35 for endogenous miRNAs and Ct<26.5 for cel-miR-257 were deemed of satisfactory quality, based on previous reports and guidelines 24–26 . All RNA samples were labeled using the same standardized protocols.…”

Improving Accuracy of Urinary miRNA Quantification in Heparinized Patients Using Heparinase I Digestion

“…This enzyme has recently been reestablished as a potential treatment to counteract RT-qPCR inhibition in the analysis of miRNA in plasma. 15,22 To develop more general standards and optimize incubation conditions for RNA isolated from urine, heparinase I was added in different concentrations during cDNA synthesis. The effect of heparinase I treatment was assessed using the PCR results from spiked-in miRNA Cel-miR-39 and the endogenous miRNAs miR-30e and miR-92a.…”

Optimized Treatment of Heparinized Blood Fractions to Make Them Suitable for Analysis

“…For example, Izraeli et al, 24 reported that 1-3 units of heparinase I for 2 hours at 25°C yielded successful PCR amplification, but no optimization of the procedure is described. More recently, Plieskatt et al 9 , described a protocol to RT-PCR amplify microRNAs isolated from heparinized blood samples using 6U of heparinase I during 1-2 h, either as a pre-treatment at 25°C or during the RT reaction at 37°C without testing whether the variables of time, temperature, or enzyme type could be optimized. An interesting alternative procedure to remove heparin from RNA preps is described by Ding et al 25 , who observed that an ultracentrifugation step prior to RNA extraction improved PCR-based amplification of viral nucleic acids from plasma.…”

“…9 However the authors did not optimize variables such as time, temperature, or performance of different types of Bacteroides heparinase. We have developed a low-cost colorimetric assay to monitor heparin removal and to determine optimal enzymatic or adsorption-dependent effective reduction of heparin levels below assay interference levels, for the processing of human blood fractions in assays affected by the presence of this anticoagulant.…”