Sep 20, 2022
RNA extraction, cDNA synthesis and Taqman qPCR
- 1UCL Institute of Neurology
Protocol Citation: gurvir.virdi 2022. RNA extraction, cDNA synthesis and Taqman qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj5rkvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 20, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70290
Keywords: ASAPCRN
Abstract
RNA extraction, cDNA synthesis and TaqMan qPCR on samples.
RNA extraction
RNA extraction
Cell pellets are snap-frozen using dry ice.
RNA is extracted using the Maxwell® RSC simply RNACells kit (Promega), and the Maxwell® RSC 48 instrument, following manufacturer instructions.
After RNA extraction, RNA concentration and quality are measured and assessed using a nanodrop.
cDNA synthesis
cDNA synthesis
Up to 1 µg of RNA per sample is reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (ThermoFisherScientific).
quantitative PCR
quantitative PCR
qPCR is performed using TaqManTM Gene Expression Assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.
For each gene, TaqManTM probes were used along with the TaqManTM master mix, and sample cDNA following the manufacturer's protocol.
Samples, along with a minus reverse transcriptase control (-RT) were ran for each gene on the QuantSudio 6 Flex Real-Time PCR System (Applied Biosystems).
The -RT served as a negative control, and the gene expression levels were normalised to the housekeeping gene GAPDH following the delta-delta Ct method. Gene expression values were expressed as the normalisation to their respective control sample.