Mar 04, 2022
RNA Extraction and RT-qPCR
- 1University of Ottawa
Protocol Citation: Haley Geertsma 2022. RNA Extraction and RT-qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.b5wmq7c6
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: March 04, 2022
Last Modified: March 04, 2022
Protocol Integer ID: 59053
Abstract
This protocol is used to extract RNA from mouse brain tissue and quantify with RT-qPCR.
Homogenize hemibrain in 3mL 1X PEPI buffer using a dounce homogenizer.
1X PEPI Buffer: 5mM EDTA + 1X protease inhibitor in 1X phosphate buffered saline (PBS)
2m
Take 3% of homogenate and add 1mL TRIzol Reagent and follow their user guide.
45m
Synthesize cDNA with 5X All-In-One RT Master Mix Kit then perform qPCR.
qPCR settings: 95oC for 5 minutes, 40x (95oC for 15 seconds then 60oC for 60 seconds), melting curve
2h