RNA Extraction and RT-qPCR
Haley Geertsma
Published: 2022-03-04 DOI: 10.17504/protocols.io.b5wmq7c6
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Abstract
This protocol is used to extract RNA from mouse brain tissue and quantify with RT-qPCR.
Steps
1.
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)
2.
Take 3% of homogenate and add 1mL TRIzol Reagent and follow their user guide.
3.
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
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