Jan 15, 2025
cDNA generation (RT) using RevertAid reverse transcriptase
- 1National Institute of Neurology and Neurosurgery
Protocol Citation: David Valle-Garcia 2025. cDNA generation (RT) using RevertAid reverse transcriptase. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjq2plx9/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: December 06, 2023
Last Modified: January 15, 2025
Protocol Integer ID: 91923
Keywords: RT, reverse transcription, RevertAid, RNA, cDNA
Disclaimer
This protocol is shared “as is” without warranty of any kind, either expressed or implied. The entire risk as to the quality and performance of the protocol is with you. Intended for basic research, only.
Abstract
This protocol is used for generating cDNA from purified RNA. It is based on RevertAid's manufacturer's protocol, with minor modifications. It can be used with other common RT enzymes such as M-MVL, just make sure to incubate the enzyme at the right temperature.
Using this protocol, we have successfully generated cDNA from eukaryotic RNA from several sources. The cDNA can be further used for qPCR, clonning, end-point PCR, etc.
Materials
50 mM Random hexamersIntegrated DNA Technologies, Inc. (IDT) RevertAid RTThermo Fisher ScientificCatalog #EP0441
10 mM dNTPsNew England BiolabsCatalog #N0447
50 uM Oligo dTIntegrated DNA Technologies, Inc. (IDT)
Safety warnings
Follow standard molecular biology safety guidelines. While handling RNA, always use clean gloves, as well as RNAse-free tips and tubes. Work on a clean area (cleaning the area with 10% bleach should get rid of most RNAses).
Before start
Make sure you have good quality RNA. You can check RNA integrity via bleach gel (see: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699176/) or using a bioanalyzer or any similar method. Ideally, you need to have 500 ng of RNA, but we have successfully generated cDNA from as little as 100 ng.
cDNA can be generated using oligo dT (for RT of poly-A RNA), random hexamers or both. Using both can increase the amount of obtained cDNA and dramatically enhance the efficiency of retro transcription if RNA is somewhat degraded. However, random hexamers should only be used if the primers used for (q)PCR are designed exon-exon and if genomic DNA contamination is not a concern. Otherwise, only use oligo dT
Before starting, thaw all components on ice, except for RevertAid, which should be taken out of the fridge only when it's needed. Briefly vortex and spin all components (except for RevertAid!) once thawed.
It is advisable to avoid repeated thaw-freeze cycles of all components. Preferably, make small aliquots of buffer, primers and dNTPs. A mix of dNTPs, oligo dT and random hexamers can also be prepared and frozen beforehand.
We usually use PCR tubes and perform all incubations in a thermocycler (PCR machine). However, using eppendorf tubes and a regular thermomixer should work as well.
Thaw RNA, RT buffer, oligo dT, and random hexamers (optional) on ice
2m
Random hexamers and oligo dT9 steps
Random hexamers plus oligo dT can increase the amount of obtained cDNA and dramatically enhance the efficiency of retro transcription if the RNA is somewhat degraded. However, they should only be used if the primers used for (q)PCR are designed exon-exon and if genomic DNA contamination is not a concern. Otherwise, only use oligo dT
Dilute 500 ng of RNA to a final volume of 12.8 µL in nuclease-free water.
Note
We use PCR tubes, but any nuclease-free sterile tube should work.
1m
Add 1 µL of 10 millimolar (mM) dTNPs
Add 1 µL of 50 micromolar (µM) oligo dT (18t)
Add 1 µL of 50 micromolar (µM) random hexamers
Note
You can also pre-mix dNTPs, oligo dT and random hexamers and add 3 µL of the mix to each RNA sample.
1m
RNA heating
RNA heating
7m
7m
This step is optional but highly advisable. Heating your RNA will avoid the formation of secondary structures that could hinder RevertAid's ability to efficiently generate cDNA from genes/regions with high GC or stem-loop structures.
Heat the prepared RNA at 50 °C for 00:05:00
Note
While the RNA is heating, you can proceed to step 5
5m
Immediately transfer all the tubes to ice and chill them for about 2 min.
Note
Make sure the tubes are cold before adding the enzyme mix!
2m
Enzyme addition
Enzyme addition
3m
3m
Per each sample, prepare the following enzyme mix:
4 µL of 5X RT buffer
0.2 µL of RevertAid
If you have more than one sample multiply by number of samples + 0.2 and mix in a single master mix. So, if you have 4 samples, multiply by 4.2. This will give you a little extra that will ensure you will not run out of enzyme master mix.
1m
Add 4.2 µL of the enzyme mix to each sample for a final volume of 20 µL
2m
Incubation
Incubation
1h 25m
1h 25m
Incubate your samples:
| A | B | C | |
| 25 °C | 10 min | Random hexamer annealing | |
| 42 °C | 60 min | Retrotranscription (if using a different enzyme to RevertAid, modify the temperature if needed) | |
| 70 °C | 15 min | Enzyme inactivation | |
| 10 °C | Hold |
1h 25m
cDNA dilution
cDNA dilution
2m
2m
Add 180 µL of nuclease-free water for a 1:10 dilution.
Optionally transfer to an eppendorf tube.
Note
Some components of the RT buffer may inhibit the efficiency of many (q)PCR polymerases. Most protocols advice not to include more than 10% of the final (q)PCR volume directly from the RT reaction. Because we always perform our qPCRs in 10ul, and we always add 1ul of cDNA, we are dangerously close to the tolerated limit. Thus, we always dilute our cDNAs with nuclease-free water and get excellent CTs and plenty of cDNA! We always recommend diluting the cDNA at least 1:2. Remember that in molecular biology, sometimes less is more.
2m
Store at -20 °C or proceed to (q)PCR.
Protocol references
RevertAid RT, thermo fisher: https://www.thermofisher.com/order/catalog/product/EP0441