TITLE: cDNA generation (RT)  using RevertAid reverse transcriptase
AUTHORS: David Valle-Garcia

[DESCRIPTION]
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.

[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.

[STEPS]
SECTION: RNA preparation
1. Thaw RNA, RT buffer, oligo dT, and random hexamers (optional) on ice

SECTION: RNA preparation
2. Dilute 500 ng of RNA to a final volume of 12.8 µL in nuclease-free water. 

 


SECTION: RNA preparation
2. Dilute 1000 ng of RNA to a final volume of 13.8 µL in nuclease-free water.
 

SECTION: RNA preparation
3. 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
 


SECTION: RNA preparation
3. Add  1 µL of 10 millimolar (mM) dTNPs 
Add  1 µL of 50 micromolar (µM) oligo dT (18t)

 

SECTION: RNA heating
4. 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 5 min 
 


SECTION: Enzyme addition
5. 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.
SECTION: RNA heating
4.1. Immediately transfer all the tubes to ice and chill them for about 2 min. 
 

SECTION: Enzyme addition
6. Add 4.2 µL of the enzyme mix to each sample for a final volume of 20 µL 

SECTION: Incubation
7. Incubate your samples:

 

SECTION: cDNA dilution
8. Add 180 µL of nuclease-free water for a 1:10 dilution. 
Optionally transfer to an eppendorf tube.

 


SECTION: RNA heating
4. 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 5 min 
 

SECTION: RNA heating
4.1. Immediately transfer all the tubes to ice and chill them for about 2 min. 
 

SECTION: Enzyme addition
5. 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.
SECTION: Enzyme addition
6. Add 4.2 µL of the enzyme mix to each sample for a final volume of 20 µL 
SECTION: Incubation
7. Incubate your samples:
 
    ABC125 °C10 minRandom hexamer annealing242 °C 60 minRetrotranscription (if using a different enzyme to RevertAid, modify the temperature if needed370 °C15 minEnzyme inactivation410 °CHold

SECTION: cDNA dilution
8. Add 180 µL of nuclease-free water for a 1:10 dilution. 
Optionally transfer to an eppendorf tube.
 

SECTION: cDNA dilution
9. Store at -20 °C or proceed to (q)PCR.
SECTION: cDNA dilution
9. Store at -20 °C or proceed to (q)PCR.