May 29, 2025
- Camille Goldman1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Camille Goldman 2025. qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl414krlo5/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: May 16, 2025
Last Modified: May 29, 2025
Protocol Integer ID: 218389
Keywords: ASAPCRN, standard protocol for rna extraction, rna extraction, rna, standard protocol
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-024297
NASA
Grant ID: 80ARC022CA004
NIH/NINDS
Grant ID: R01NS114239
NIH/NINDS
Grant ID: UH3NS115064
NIN/NIA
Grant ID: T32AG04968
NIH/NINDS
Grant ID: F31NS13090
New York State Department of Health
Grant ID: NYSTEM-C32561GG
Abstract
Standard protocol for RNA extraction and qPCR
Troubleshooting
RNA Extraction
Wash cells with PBS
Lyse cells in TRIzol (Invitrogen 15596018). For 1 well of a 6-well plate, use 300 uL of TRIzol
Freeze TRIzol lysate or continue to the next step
Use an RNA extraction kit, following manufacturer's recommendation (eg Zymo R2062)
Measure extracted RNA concentration and purity on a spectrophotometer
Store RNA at -80°C
cDNA
Thaw RNA on ice if not continuing directly from step 5
Prepare the reverse transcription master mix as follows: 2X RT buffer (Thermo Scientific EP0752), 1mM each dNTP mix (Thermo Scientific #R0191), 10uM random hexamers (Thermo Scientific #SO142), 50 U Maxima H Minus Reverse Transcriptase per reaction(Thermo Scientific EP0752), and nuclease free water up to 10uL per reaction.
In a PCR tube, add 1 ug of RNA and nuclease free water up to 10 uL. The amount of RNA can be lowered if RNA concentration is low.
Add 10 uL of the master mix from step 8 to each sample
Incubate for 10 minutes at 25°C
Incubate for 30 minutes at 50°C
Store cDNA at -20°C
qPCR
Thaw cDNA on ice if not continuing directly from step 12
Dilute cDNA 1:10 in nuclease free water
Prepare primer mixes with 10uM of forward primer and 10uM of reverse primer in nuclease free water
In each well of a 384 well plate, combine 2X PowerUp SYBR Green Master Mix (Applied Biosystems A25741), 1uM each of forward and reverse primers, and diluted cDNA to a final volume of 10uL.
Run qPCR with the following cycle:
2 minutes at 50°C
10 minutes at 95°C
40 cycles of 15 seconds at 95°C and 1 minute at 60°C
Optionally, include a melting curve stage: 15 seconds at °C, 1 minute at 60°C and a gradual increase of 0.05°C/second back to 95°C
Analyze using the ΔΔCt method