Aug 09, 2023

qRT-PCR sample preparation

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l22713l1y/v1
  • 1Lazarou Lab, WEHI
Louise Uoselis
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l22713l1y/v1
Protocol Citation: Louise Uoselis 2023. qRT-PCR sample preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22713l1y/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: August 09, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 86219
Keywords: ASAPCRN, pcr sample preparation, pcr sample preparation protocol, pcr sample preparation for analysis, rotorgeneq machine, qiagen
Abstract
Protocol for qRT-PCR sample preparation for analysis using a RotorGeneQ machine (Qiagen).
Synthesise cDNA libraries from total RNA from each sample using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), using Oligo(dT)20 primers.
Place the synthesised libraries on ice, and dilute the libraries 1:3 by adding 60 µL of DEPC-treated H2O to each sample, pipetting up and down gently to mix ~ 8 times.

Dilute primer stocks by adding 2 µL of the forward primer, and 2 µL of the reverse primer, to 46 µL of DEPC-treated H2O on ice to make a final combined forward and reverse primer stock of 4 micromolar (µM) forward primer and 4 micromolar (µM) reverse primer for each gene target to be analysed.

Thaw the 2x QuantiNova SYBR green master mix (Qiagen) on ice.
Depending on the number of samples being analysed, assemble a sample master mix (not containing the cDNA library) containing the following:


ReagentVolume (1x)
2x SYBR Green Master Mix5 uL
4 uM forward and 4 uM reverse primer master mix1 uL
cDNA library (to be added individually to each tube)4 uL
Total volume10 uL

To qRT-PCR tubes sitting on ice (Qiagen – RotorGeneQ compatible), add 6 µL of the master mix for each target gene to the desired tubes, and add 4 µL of the appropriate cDNA library to each sample.
Tap the rack holder to ensure all solution is at the bottom of the tube before capping each sample.(NOTE: samples do not need to be mixed prior to analysis)
Load and run the samples on a RotorGeneQ (Qiagen) machine using the desired parameters for the gene targets you are analysing.