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Circumventing qPCR inhibition to amplify miRNAs in plasma.

ABSTRACT: BACKGROUND:Circulating 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. FINDINGS:Treatment 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. CONCLUSION:Incorporating Bacteroides heparinase I treatment into conventional RT protocols targeting c-miRNA in plasma can be expected to expedite the discovery of biomarkers.

SUBMITTER: Plieskatt JL

PROVIDER: S-EPMC4114091 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Circumventing qPCR inhibition to amplify miRNAs in plasma.

Plieskatt Jordan L JL Feng Yanjun Y Rinaldi Gabriel G Mulvenna Jason P JP Bethony Jeffrey M JM Brindley Paul J PJ

Biomarker research 20140722

<h4>Background</h4>Circulating 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 ...[more]

PMID: 25075309

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Project description:BackgroundMicroRNAs (miRNAs) are noncoding RNAs that are highly relevant as disease biomarkers. Several studies that explored the role of miRNAs in Alzheimer's disease (AD) demonstrated their usefulness in clinical identification. Nevertheless, miRNAs that may act as endogenous controls (ECs) have not yet been established. The identification of ECs would contribute to the standardization of these biomarkers in AD. The objective of the study was to identify miRNAs that can be used as ECs in AD.MethodsWe evaluated 145 patients divided into two different cohorts. One was a discovery cohort of 19 women diagnosed with mild to moderate AD (Mini-Mental State Examination (MMSE) score ≥ 20) and with confirmed pathologic levels of Aβ42 in CSF. The stability assessment cohort consisted of 126 individuals: 24 subjects without AD or any kind of dementia and negative for all core CSF biomarkers of AD, 25 subjects with MCI and negative for CSF biomarkers (MCI -), 22 subjects with MCI and positive for CSF biomarkers (MCI +), and 55 subjects with AD and positive for CSF biomarkers. In the discovery cohort, a profile of 384 miRNAs was determined in the plasma by TaqMan low-density array. The best EC candidates were identified by mean-centering and concordance correlation restricted normalization methods. The stability of the EC candidates was assessed using the GeNorm, BestKeeper, and NormFinder algorithms.ResultsNine miRNAs (hsa-miR-324-5p, hsa-miR-22-5p, hsa-miR-103a-2-5p, hsa-miR-362-5p, hsa-miR-425-3p, hsa-miR-423-5p, hsa-let-7i-3p, hsa-miR-532-5p, and hsa-miR-1301-3p) were identified as EC candidates in the discovery cohort. The validation results indicated that hsa-miR-103a-2-5p was the best EC, followed by hsa-miR-22-5p, hsa-miR-1301-3p, and hsa-miR-425-3p, which had similar stability values in all three algorithms.ConclusionsWe identified a profile of four miRNAs as potential plasma ECs to be used for normalization of miRNA expression data in studies of subjects with cognitive impairment.

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Circumventing qPCR inhibition to amplify miRNAs in plasma.

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Circumventing qPCR inhibition to amplify miRNAs in plasma.

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