Quantification of microRNA in plasma using probe based TaqMan assays

Is microRNA purification required?

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

*Kontaktforfatter for dette arbejde

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Resumé

Objective: Circulating microRNAs are promising diagnostics and prognostics biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge, which in part may be due to preanalytical factors. MicroRNA purification has been identified as the major contributor to the total intra assay variation, thus we found great interest in recent papers describing methods for direct quantification of circulating microRNAs without the purification step. With one exception, all the studies we identified where a direct quantification of circulating microRNAs had been performed were using SYBR Green chemistry. In our laboratory we use platelet-poor plasma and TaqMan assays for microRNA analysis, and thus we investigated whether we could adapt the procedures for the direct reverse transcription described by these studies to be used with our TaqMan assays. Results: We did not achieve valid results by direct quantification of selected microRNAs (miR-92a, miR-16 and miR-126) in platelet-poor plasma using TaqMan assays.

OriginalsprogEngelsk
Artikelnummer261
TidsskriftBMC Research Notes
Vol/bind12
Antal sider5
ISSN1756-0500
DOI
StatusUdgivet - 10. maj 2019

Fingeraftryk

Plasma probes
MicroRNAs
Purification
Assays
Platelets
Plasmas
Biomarkers
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title = "Quantification of microRNA in plasma using probe based TaqMan assays: Is microRNA purification required?",
abstract = "Objective: Circulating microRNAs are promising diagnostics and prognostics biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge, which in part may be due to preanalytical factors. MicroRNA purification has been identified as the major contributor to the total intra assay variation, thus we found great interest in recent papers describing methods for direct quantification of circulating microRNAs without the purification step. With one exception, all the studies we identified where a direct quantification of circulating microRNAs had been performed were using SYBR Green chemistry. In our laboratory we use platelet-poor plasma and TaqMan assays for microRNA analysis, and thus we investigated whether we could adapt the procedures for the direct reverse transcription described by these studies to be used with our TaqMan assays. Results: We did not achieve valid results by direct quantification of selected microRNAs (miR-92a, miR-16 and miR-126) in platelet-poor plasma using TaqMan assays.",
keywords = "Direct plasma RT-qPCR, MicroRNA, Sample preparation, TaqMan assays",
author = "Binderup, {Helle Glud} and Madsen, {Jonna Skov} and Brasen, {Claus Lohman} and Kim Houlind and Andersen, {Rikke Fredslund}",
year = "2019",
month = "5",
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doi = "10.1186/s13104-019-4301-5",
language = "English",
volume = "12",
journal = "BMC Research Notes",
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Quantification of microRNA in plasma using probe based TaqMan assays : Is microRNA purification required? / Binderup, Helle Glud; Madsen, Jonna Skov; Brasen, Claus Lohman; Houlind, Kim; Andersen, Rikke Fredslund.

I: BMC Research Notes, Bind 12, 261, 10.05.2019.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Quantification of microRNA in plasma using probe based TaqMan assays

T2 - Is microRNA purification required?

AU - Binderup, Helle Glud

AU - Madsen, Jonna Skov

AU - Brasen, Claus Lohman

AU - Houlind, Kim

AU - Andersen, Rikke Fredslund

PY - 2019/5/10

Y1 - 2019/5/10

N2 - Objective: Circulating microRNAs are promising diagnostics and prognostics biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge, which in part may be due to preanalytical factors. MicroRNA purification has been identified as the major contributor to the total intra assay variation, thus we found great interest in recent papers describing methods for direct quantification of circulating microRNAs without the purification step. With one exception, all the studies we identified where a direct quantification of circulating microRNAs had been performed were using SYBR Green chemistry. In our laboratory we use platelet-poor plasma and TaqMan assays for microRNA analysis, and thus we investigated whether we could adapt the procedures for the direct reverse transcription described by these studies to be used with our TaqMan assays. Results: We did not achieve valid results by direct quantification of selected microRNAs (miR-92a, miR-16 and miR-126) in platelet-poor plasma using TaqMan assays.

AB - Objective: Circulating microRNAs are promising diagnostics and prognostics biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge, which in part may be due to preanalytical factors. MicroRNA purification has been identified as the major contributor to the total intra assay variation, thus we found great interest in recent papers describing methods for direct quantification of circulating microRNAs without the purification step. With one exception, all the studies we identified where a direct quantification of circulating microRNAs had been performed were using SYBR Green chemistry. In our laboratory we use platelet-poor plasma and TaqMan assays for microRNA analysis, and thus we investigated whether we could adapt the procedures for the direct reverse transcription described by these studies to be used with our TaqMan assays. Results: We did not achieve valid results by direct quantification of selected microRNAs (miR-92a, miR-16 and miR-126) in platelet-poor plasma using TaqMan assays.

KW - Direct plasma RT-qPCR

KW - MicroRNA

KW - Sample preparation

KW - TaqMan assays

U2 - 10.1186/s13104-019-4301-5

DO - 10.1186/s13104-019-4301-5

M3 - Journal article

VL - 12

JO - BMC Research Notes

JF - BMC Research Notes

SN - 1756-0500

M1 - 261

ER -