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Quantification of microRNA in plasma using probe based TaqMan assays: is microRNA purification required?

Author(s): Binderup HG, Madsen JS, Brasen CL, Houlind K, Andersen RF

Publication: BMC Res Notes, 2019, Vol. 12, Page 261

PubMed ID: 31077242 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated if miRNA in plasma, PPP, or serum could be quantified after a denaturation step with or without heating, RNAse inhibitor addition, and centrifugation instead of miRNA extraction. Blood was collected from 10 healthy volunteers (collection details not specified). EDTA plasma and serum were obtained by centrifugation of the blood at 2000 x g for 10 min and platelet-poor plasma (PPP) by centrifugation twice at 3000 x g for 15 min. To inactivate proteins, plasma was diluted 1:1 in a buffer containing 2.5% Tween-20, 50 mM Tris–HCl, and 1 mM EDTA without pH adjustment or adjusted to pH 8.0. After denaturation, the specimen was either directly used for reverse transcription, heated and centrifuged (75˚C or 70 ˚C for 10 min followed by chilling on ice and centrifugation at 10,000 x g for 10 min at 4˚C) prior to reverse transcription, or heated at 70˚C for 10 min with no centrifugation prior to reverse transcription. RNAse inhibitor was added to specimens incubated at 70˚C before the heating step. Cel-miR-39 was added to all specimens before or after heating. RNA was also extracted directly from PPP using the NucleoSpin miRNA plasma kit. Specimens were reverse transcribed using the TaqMan MicroRNA Reverse Transcription kit. Specimens not subjected to centrifugation after denaturation were centrifuged for 10 min at 10,000 x g after reverse transcription. miRNAs were quantified by real-time PCR using TaqMan assays for miR-92a, miR-126, miR-16, and cel-miR-39.

   Summary of Findings:

   Overall, results were comparable between specimens denatured with unadjusted pH and those with pH adjusted to 8.0. When the specimen was mixed with denaturation buffer and not heated or centrifuged until after reverse transcription or was heated to 75˚C for 10 min followed by centrifugation at 10,000 x g for 10 min, all CT values were above 35. While cel-mIR-39, miR-126, and miR-16 were not quantifiable or had CT values >35, miR-92a produced CT values of 31-34 and 33-35 in the plasma and PPP specimens, respectively in specimens to which RNAse inhibitor was added prior to incubation at 70˚C for 10 min and centrifugation at 10,000 x g,. cel-miR-39 CT values of 25-27 and target miRNA CT values of 28-40 were observed for specimens to which RNAse inhibitor was added prior to incubation at 70˚C for 10 min and centrifuged after reverse transcription, regardless of whether the specimen was PPP or plasma. The authors reported no differences in CT values between plasma and PPP, indicating the presence of some inhibitors in this method. Additionally, CT values were still approximately 4-19 CT higher for these specimens than for specimens from which RNA was extracted using NucleoSpin miRNA plasma kit.

   Since plasma and PPP produced comparable CT values, the authors performed a dilution experiment to determine if this method results in inhibition. Dilution of PPP 10-fold produced the expected increase in CT values for miR-92a and miR-126 but not miR-16. Further dilution (100-fold) did not result in further increases in the CT values. Dilution of serum specimens produced inconsistent results. Interestingly, addition of the buffer to purified miRNA had no effect on the CT values, indicating that it is not an inhibitory effect of the buffer components. Thus the authors conclude this method is not suitable for quantification of miRNA.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Serum

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Platelet-poor plasma Serum
   Analyte Extraction and Purification	Analyte isolation method	Extracted with NucleoSpin miRNA plasma kit Denatured only
   Analyte Extraction and Purification	RNase inactivation	RNAse inhibitor added No RNAse inhibitor added
   Analyte Extraction and Purification	Incubation duration/condition	75˚C for 10 min 70˚C for 10 min
   Real-time qRT-PCR Specific	Template/input amount	Not diluted Diluted 1:10 Diluted 1:100
   Real-time qRT-PCR Specific	Targeted nucleic acid	cel-miR-39 miR-92a miR-126 miR-16

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