MicroRNA profiling in plasma samples using qPCR arrays: Recommendations for correct analysis and interpretation

PloS One
Andreas B GevaertAmaryllis H Van Craenenbroeck

Abstract

MicroRNA (miRNA) regulate gene expression through posttranscriptional mRNA degradation or suppression of translation. Many (pre)analytical issues remain to be resolved for miRNA screening with TaqMan Low Density Arrays (TLDA) in plasma samples, such as optimal RNA isolation, preamplification and data normalization. We optimized the TLDA protocol using three RNA isolation protocols and preamplification dilutions. By using 100μL elution volume during RNA isolation and adding a preamplification step without dilution, 49% of wells were amplified. Informative target miRNA were defined as having quantification cycle values ≤35 in at least 20% of samples and low technical variability (CV across 2 duplicates of 1 sample <4%). A total of 218 miRNA was considered informative (= 59% of all target miRNA). Different normalization strategies were compared: exogenous Ath-miR-159a, endogenous RNA U6, and three mathematical normalization techniques: geNorm (Qbase, QB) and NormFinder (NF) normalization algorithms, and global mean calculation. To select the best normalization method, technical variability, biological variability, stability, and the extent to which the normalization method reduces data dispersion were calculated. The geNorm normal...Continue Reading

References

Oct 23, 2008·Nucleic Acids Research·Pieter MestdaghJo Vandesompele
Jan 30, 2009·RNA·Sylvain PradervandKeith Harshman
Mar 18, 2009·Laboratory Investigation; a Journal of Technical Methods and Pathology·Angela B Y HuiFei-Fei Liu
Jun 18, 2009·Genome Biology·Pieter MestdaghJo Vandesompele
Feb 12, 2010·Methods : a Companion to Methods in Enzymology·Evan M KrohMuneesh Tewari
Jul 29, 2010·British Journal of Cancer·I Van der AuweraS J Van Laere
Jan 21, 2011·Nature·Eric M Small, Eric N Olson
Apr 14, 2011·Clinical Chemistry·Jennifer S McDonaldAlicia Algeciras-Schimnich
Nov 16, 2011·Journal of Bioinformatics and Computational Biology·Ameya DeoAngelica Lindlöf
Dec 7, 2011·Methods in Molecular Biology·Barbara D'haeneJo Vandesompele
Jan 21, 2012·Blood·Willem Stoorvogel
Feb 14, 2012·Nature Cell Biology·Eduard HergenreiderStefanie Dimmeler
Apr 18, 2014·Epigenetics : Official Journal of the DNA Methylation Society·Veronica J Peschansky, Claes Wahlestedt
Apr 19, 2015·BMC Molecular Biology·Vlasta KorenkováRobert Sjöback
Sep 27, 2015·Clinical Chemistry·Heidi SchwarzenbachKlaus Pantel
Oct 18, 2015·American Journal of Physiology. Heart and Circulatory Physiology·Amaryllis H Van CraenenbroeckEmeline M Van Craenenbroeck
Feb 9, 2016·Expert Review of Molecular Diagnostics·Margaret Dellett, David Arthur Simpson
Feb 13, 2016·European Journal of Heart Failure·Eline L VegterAdriaan A Voors
Apr 15, 2016·Biomolecular Detection and Quantification·Steven T OkinoYan Wang
Aug 20, 2016·Nephrology, Dialysis, Transplantation : Official Publication of the European Dialysis and Transplant Association - European Renal Association·Amaryllis H Van CraenenbroeckMarie M Couttenye
Feb 6, 2017·Annals of Translational Medicine·Maurice W J de RondeSara-Joan Pinto-Sietsma
Feb 18, 2017·Nature Reviews. Drug Discovery·Rajesha Rupaimoole, Frank J Slack

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Citations

Sep 15, 2018·Current Protocols in Human Genetics·Isabel WitvrouwenAmaryllis H Van Craenenbroeck
Jan 4, 2019·Clinical Journal of the American Society of Nephrology : CJASN·Kristien J LedeganckAmaryllis H Van Craenenbroeck
Jan 23, 2020·Journal of Cellular and Molecular Medicine·Taís Silveira AssmannJ Alfredo Martínez
Apr 9, 2020·Epigenomics·Irena AbramovicNino Sincic
Jul 8, 2020·Chronobiology International·Taís Silveira AssmannJ Alfredo Martínez
Jun 18, 2020·Frontiers in Oncology·Ruma Dey GhoshSusanta Roychoudhury
Nov 16, 2018·Molecular Neurobiology·Theresa A LusardiJulie A Saugstad
Sep 22, 2020·Journal of Alzheimer's Disease : JAD·Ursula S SandauJulie A Saugstad
Dec 12, 2019·Frontiers in Molecular Neuroscience·Theodora KalpachidouMichaela Kress
Aug 6, 2020·Adipocyte·Helene A FachimAdrian H Heald
Nov 11, 2020·Molecular Biology Reports·Jesse D ArmitageDino B A Tan
Dec 18, 2020·International Journal of Molecular Sciences·Taís Silveira AssmannJ Alfredo Martínez
Mar 21, 2021·European Journal of Preventive Cardiology·Isabel WitvrouwenEmeline M Van Craenenbroeck
May 28, 2021·Physiological Reports·Miroslava RabajdovaAndrea Madarasova Geckova
Sep 12, 2021·European Journal of Preventive Cardiology·Andreas B GevaertUNKNOWN OptimEx-Clin Study Group

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Methods Mentioned

BETA
Low Density Arrays
Low
PCR

Software Mentioned

package
R
SDS
geNorm
NormqPCR package
TLDA
NormFinder
irr
Qbase

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