Jul 10, 2024
mRNA extraction and cDNA preparation
- 1Duke University
Protocol Citation: Shiyi Wang 2024. mRNA extraction and cDNA preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l623n5gqe/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 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103172
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
mRNA extraction and cDNA preparation
**Thaw and Resuspend Cells** - Thaw cells stored in TRIzol (15596026, Invitrogen). - Resuspend cells in 1 mL of TRIzol.
**Add Chloroform** - Add 200 μL of chloroform to the samples.
**Centrifuge Samples** - Centrifuge the samples at 12,000 g for 15 minutes at 4°C.
**Collect Aqueous Phase** - Carefully collect the aqueous phase.
**Precipitate RNA** - Add GlycoBlue Coprecipitant (AM9515, Invitrogen) and isopropanol to precipitate the RNA.
**Wash RNA Pellet** - Wash the RNA pellet with 75% ethanol.
**Dry and Resuspend RNA** - Air-dry the RNA pellet. - Resuspend the RNA in 40 μL of nuclease-free water.
**Isolate RNA** - Isolate the RNA using the Zymo Research RNA Clean & Concentrator-5 Kit (R1014, Zymo).
**Quantify RNA** - Quantify the RNA using the Qubit RNA HS Assay Kit (Q32852, Invitrogen).
**Equalize RNA Concentrations** - Dilute the RNA to equalize concentrations.
**Generate cDNA Libraries** - Use the qScript cDNA SuperMix (101414-102, VWR) for cDNA library preparation. - Follow this temperature profile: - 25°C for 5 minutes - 42°C for 30 minutes - 85°C for 5 minutes
**Dilute and Store cDNA** - Dilute the resulting cDNA threefold. - Store the cDNA at −80°C.