Apr 18, 2022
Relative quantification of mRNA transcript levels by qPCR
- Will Hancock-Cerutti1,2,3,
- Zheng Wu4,5,
- Gerald S. Shadel5,
- Pietro De Camilli1,3
- 1Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Interdisciplinary Neuroscience Program and MD-PhD Program, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 4Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 5Salk Institute for Biological Studies, La Jolla, CA, USA
Protocol Citation: Will Hancock-Cerutti, Zheng Wu, Gerald S. Shadel, Pietro De Camilli 2022. Relative quantification of mRNA transcript levels by qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnz12qg5d/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: July 08, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 51403
Keywords: RNA, cDNA, qPCR, ASAPCRN
Abstract
This method describes isolation of RNA from cultured cells, generation of cDNA, and relative quantification of transcript levels by qPCR.
Attachments
dxwybkjz7.pdf
119KB
Materials
Solutions to prepare:
DMEM solution:
| FBS | 10% | |
| Penicillin | 100 U/ml | |
| Streptomycin | 100 mg/ml | |
| L-glutamine | 2 mM |
Cell culture and treatments
Cell culture and treatments
3d
3d
Culture the HeLa-M cells at 37 °C in 5% CO2 and DMEM containing 10% FBS, 100 U/ml penicillin, 100 mg/mL streptomycin, and 2 millimolar (mM) L-glutamine (all from Gibco).
For any given experiment, plate the cells at such density so as to be approximately 90% confluent at the time of lysis.
For experiments using siRNA, transfect 60 pmols of the indicated siRNA using 6 µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol. Lyse the cells 72:00:00 after siRNA transfection.
3d
Cell lysis, RNA purification, and qPCR
Cell lysis, RNA purification, and qPCR
Aspirate media from cells and rinse cells with PBS On ice .
Isolate RNA using RNeasy Micro Plus kit (Qiagen) according to manufacturer’s protocol.
Generate cDNA from 1 µg purified RNA using iScript cDNA synthesis Kit (Bio-Rad) according to manufacturer’s protocol.
Dilute the iScript reaction to a total of 400 µL Sterile Water (American Bio).
Combine 10 µL SYBR Green Master Mix (BioRad) with 6.78 µL Sterile Water (American Bio) per sample.
Combine 16.78 µL diluted SYBR Green Master Mix with 0.61 µL each of 10 micromolar (µM) forward and reverse primers per sample. Pipette this mixture into wells of 96-well qPCR plate. Perform at least two technical replicates for each sample.
Pipette 2 µL of diluted RNA from step 7 in well with SYBR Green Master Mix.
Cover plate with Optical Adhesive Covers (Applied Biosystems).
Spin down plate in table top centrifuge.
Run qPCR in CFX96 Real-Time System (BioRad) using the following protocol:
| A | B | C | |
| 95 ˚C | 3 min | ||
| 95 ˚C | 10 sec | Repeat 39x | |
| 55 ˚C | 10 sec | ||
| 72˚C | 30 sec | ||
| 95 ˚C | 10 sec | ||
| 65 ˚C | 5 sec | ||
| 95 ˚C | 5 sec |
Data analysis
Data analysis
Subtract the housekeeping gene (b-actin) mean threshhold cycle (Ct) values from transcript of interest mean Ct values to calculate ΔCt.
Subtract the ΔCt of the control sample from each sample ΔCt to calculate the ΔΔCt value.
Calculate relative expression using the 2−ΔΔCt method.