Apr 20, 2026

RNA isolation and cDNA synthesis

 Forked from mRNA extraction and cDNA preparation
DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6dr6rvqe/v1
  • 1Duke University;
  • 2KU Leuven;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6dr6rvqe/v1
Protocol Citation: Shiyi Wang, Sarah van Veen 2026. RNA isolation and cDNA synthesis. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dr6rvqe/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: January 09, 2025
Last Modified: April 20, 2026
Protocol  Integer ID: 117972
Keywords: ASAPCRN, RNA isolation, cDNA synthesis, primary astrocytes, primary neurons, isolation of rna, rna isolation, subsequent cdna synthesis for downstream application, cdna synthesis this protocol detail, subsequent cdna synthesis, cdna synthesis, rna, qpcr, such as qpcr, purification, isolation
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
This protocol details the isolation of RNA, purification, and subsequent cDNA synthesis for downstream applications such as qPCR.
Materials
  • Cell lysates stored in TRIzol
  • TRIzol (Invitrogen, Cat# 15596026) 
  • GlycoBlue Coprecipitant (2 μL of 15 mg mL−1; Invitrogen, Cat# AM9515)
  • DNase I Set (RNase-free)(Zymo Research, Cat# E1010)
  • RNA Clean & Concentrator-5 Kit (Zymo Research, Cat# R1014) 
  • qScriptTM cDNA SuperMix (VWR, Cat# 101414-102)
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
RNA isolation
1h
Thaw cells stored in TRIzol to Room temperature .
Resuspend cells in 800 µL of TRIzol.
Add 200 µL of chloroform to the samples and invert to mix.

Centrifuge the samples at 12000 x g, 4°C, 00:15:00 .

15m
Carefully collect the aqueous phase (top clear layer) and dispense into a 1.5 ml RNase free eppendorf tube.

Note
Be careful not to disturb the interphase!

Add 2 µL GlycoBlue Coprecipitant and 500 µL isopropanol to precipitate the RNA. Incubate for 00:10:00 .

10m
Centrifuge the samples at 12000 x g, 4°C, 00:10:00 .
10m
Carefully aspirate the TRIzol. 
Wash the RNA pellet with 1 mL of 75% ethanol.

Centrifuge the samples at 7500 x g, 4°C, 00:05:00 .
5m
Air-dry the RNA pellet for 00:05:00 .

5m
Resuspend the RNA in 40 µL of nuclease-free water.

DNaseI treatment
1h
Add 5 µL of Zymosan DNaseI and 5 µL of DNA digestion buffer to the 40 µL of RNA.

Incubate at Room temperature for 00:15:00 .

15m
RNA clean-up and concentration
3m 30s
Isolate the RNA using the Zymo Research RNA Clean & Concentrator-5 kit.
Add 2 volumes (100 µL ) RNA binding buffer to each sample and mix.
Add equal volume (150 µL ) of 200-proof 100% ethanol and mix.
Transfer sample to column. Centrifuge at 16000 rcf for 00:00:30 . Discard flowthrough.
30s
Add 400 µL RNA Prep buffer to column and centrifuge at 16000 rcf for 00:00:30 . Discard flowthrough.
30s
Add 700 µL RNA Wash buffer to column and centrifuge at 16000 rcf for 00:02:00 . Discard flowthrough.
2m
Transfer column to nuclease-free tube and add 6 µL of UP-water directly to column. Centrifuge at 16000 rcf for 00:00:30 .

30s
Measure RNA concentration using Nanodrop.
cDNA synthesis
40m
Use the qScript cDNA SuperMix for cDNA library preparation.
Combine: 
cDNA SuperMix (5X)2 uL
RNA template1 pg to 1 ug
nuclease-free waterFill to 10 uL final Rxn volume

Incubate using the following rxn:
  • 00:05:00 at 25 °C
  • 00:30:00 at 42 °C
  • 00:05:00 at 85 °C
40m
Add 20 µL of ultra pure water to the 10 µL cDNA reaction. Pipette up and down 10 times to thoroughly mix.  

Aliquot your samples into labeled PCR tubes (~10uL per tube).
Store samples at -80 °C until ready for qPCR.