Systematic evaluation of RT-qPCR kits and protocols for detection and quantitation of microRNAs

Abstract

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs with an average length of ~ 22 nucleotides. The main function of miRNA is post-transcriptional regulation of gene expression by targeting specific mRNAs. The value of miRNA regulation and expression has become an interesting focus in diagnostics. Extracellular miRNAs have been found in a variety of body fluids, making miRNAs suitable as noninvasive biomarkers. For the understanding of miRNAs biological roles and for identifying potential biomarkers, reliable technologies are required for detection and quantitation. Reverse transcription of miRNA to cDNA template followed by quantitative PCR (RT-qPCR) are often the method of choice, however different RT-qPCR technologies have been observed to give varying results.

In this thesis, a systematic evaluation has been conducted for three kits and protocols to perform RT-qPCR for detection and quantitation of miRNA. The three different methods evaluated was miRCURY RT-qPCR setup (Qiagen), TaqMan Advanced RT-qPCR setup (Thermo Fisher Scientific) with and without preamplification, and TaqMan MicroRNA RT- qPCR setup (Thermo Fisher Scientific) with and without uracil-N glycosylase (UNG) included in the master mix. All setups included assays for six miRNAs represented by synthetic material. The miRNAs were analyzed once with added synthetical noise and once with added biological noise.

The objective of the evaluation was to find a preferred RT-qPCR setup that later can be used for verifying miRNA-findings from RNA sequencing. It was indicated that miRCURY RT- qPCR setup gave best result for verifying lowly expressed miRNAs. This was based on least deviation in the reaction efficiency, low indication of false positives and unspecific amplification and a wide range of input of total RNA. This study indicated that TaqMan Advanced RT-qPCR setup without the preamplification reaction, should not be used for detection and quantitation of lowly expressed miRNA. This was based on the setup’s poor reaction efficiency, observed detection range and precision. TaqMan MicroRNA RT-qPCR setup indicated being advantageous when few miRNA-targets and replicates are required due to the lowest price per reaction for one target and the feasibility. By including UNG in the master mix the deviation of the reaction efficiency decreased.

MicroRNA (miRNA) er korte, ikke-kodene, enkeltrådige RNA med en gjennomsnittlig størrelse på ~ 22 nukleotider. Hovedfunksjonen til miRNA er regulering av genuttrykk etter transkripsjonen. Dette gjøres ved at miRNA bindes til komplementære sekvenser på målmRNA. Genregulering av miRNA har fått økende fokus i sykdomssammenheng. MicroRNA har blitt funnet i ulike kroppsvæsker, blant annet i plasma, og dette gjør miRNA egnet som ikke-invasive biomarkører. For å identifisere potensielle biomarkører er det nødvendig med nøyaktige metoder til deteksjon og kvantifisering. Reversert transkripsjon av miRNA til cDNA, etterfulgt av kvantitativ PCR (RT-qPCR) er ofte den foretrukne metoden, men ulike RT-qPCR fremgangsmåter har vist varierende resultat.

I denne studien ble det utført en systematisk evaluering av tre ulike fremgangsmåter for å utføre RT-qPCR til deteksjon og kvantifisering av miRNA. De tre ulike fremgangsmåtene som ble vurdert var miRCURY RT-qPCR oppsett (Qiagen), TaqMan Advanced RT-qPCR oppsett (Thermo Fisher Scientific) med og uten pre-amplifisering og TaqMan MicroRNA RTqPCR oppsett (Thermo Fisher Scientific) med og uten uracil-N glycosylase (UNG) inkludet i reaksjonsmiksen. Vurderingen ble utført med assay for seks miRNA representert av syntetisk materiale. MiRNAene ble analysert både med inkludert syntetisk og biologisk bakgrunnsstøy i to ulike oppsett.