Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions

Helle Glud Binderup*, Jonna Skov Madsen, Niels Henrik Helweg Heegaard, Kim Houlind, Rikke Fredslund Andersen, Claus Lohman Brasen

*Kontaktforfatter for dette arbejde

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Resumé

Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8% and 10.5–31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1%, and the inter-assay variation 5.7–26.7%. Plasma preparation and microRNA purification were found to account for 39–73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.

OriginalsprogEngelsk
Artikelnummere0201069
TidsskriftPLOS ONE
Vol/bind13
Udgave nummer7
Antal sider13
ISSN1932-6203
DOI
StatusUdgivet - 2018

Fingeraftryk

MicroRNAs
microRNA
Plasmas
Assays
assays
Biomarkers
Platelets
droplets
Real-Time Polymerase Chain Reaction
biomarkers
quantitative polymerase chain reaction
Polymerase Chain Reaction
Centrifugation
Validation Studies
reproducibility
centrifugation
Purification
Health

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title = "Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions",
abstract = "Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8{\%} and 10.5–31.4{\%}, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1{\%}, and the inter-assay variation 5.7–26.7{\%}. Plasma preparation and microRNA purification were found to account for 39–73{\%} of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.",
keywords = "Biomarkers/blood, Blood Platelets, Centrifugation, Humans, MicroRNAs/blood, Molecular Diagnostic Techniques, Plasma, Polymerase Chain Reaction, Pre-Analytical Phase",
author = "Binderup, {Helle Glud} and Madsen, {Jonna Skov} and {Helweg Heegaard}, {Niels Henrik} and Kim Houlind and Andersen, {Rikke Fredslund} and Brasen, {Claus Lohman}",
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language = "English",
volume = "13",
journal = "P L o S One",
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Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions. / Binderup, Helle Glud; Madsen, Jonna Skov; Helweg Heegaard, Niels Henrik; Houlind, Kim; Andersen, Rikke Fredslund; Brasen, Claus Lohman.

I: PLOS ONE, Bind 13, Nr. 7, e0201069, 2018.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions

AU - Binderup, Helle Glud

AU - Madsen, Jonna Skov

AU - Helweg Heegaard, Niels Henrik

AU - Houlind, Kim

AU - Andersen, Rikke Fredslund

AU - Brasen, Claus Lohman

PY - 2018

Y1 - 2018

N2 - Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8% and 10.5–31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1%, and the inter-assay variation 5.7–26.7%. Plasma preparation and microRNA purification were found to account for 39–73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.

AB - Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8% and 10.5–31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1%, and the inter-assay variation 5.7–26.7%. Plasma preparation and microRNA purification were found to account for 39–73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.

KW - Biomarkers/blood

KW - Blood Platelets

KW - Centrifugation

KW - Humans

KW - MicroRNAs/blood

KW - Molecular Diagnostic Techniques

KW - Plasma

KW - Polymerase Chain Reaction

KW - Pre-Analytical Phase

U2 - 10.1371/journal.pone.0201069

DO - 10.1371/journal.pone.0201069

M3 - Journal article

VL - 13

JO - P L o S One

JF - P L o S One

SN - 1932-6203

IS - 7

M1 - e0201069

ER -