Mike Barnkob:Protocols/Cloning/cDNA library from mRNA
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Creating cDNA from mRNA
Protocol on how to convert mRNA into cDNA.
General notes
Lab practice:
- Wear a clean lab coat.
- Change gloves vigorously.
- Use dedicated (or at least very clean) pipets.
- Clean lab bench (it probably needs it too) with 1% SDS and rinse with 70% ethanol.
- Soak all utilities in 1% SDS overnight and rinse with 70% ethanol.
- Do not use microcentrifuge that is also being used for minipreps.
Experimental setup:
- Use two types on negative control for experiment:
- One negative consisting of a duplicate sample but without the RT enzyme added.
- One negative consisting of the RT enzyme and master mix but with nuclease free water, instead of mRNA (so no mRNA in this sample).
- Use 500-1000ng RNA for each sample.
Oligo(dT) or Random primers?
- Oligo(dT)'s will amplify targets with poly-adenolated tails and is good for construction of cDNA libraries since this will increase the chance of getting rare targets.
- For RT-PCR random primers will be fine, since most PCR primers will amplify even rare targets.
Applied Biosystem High Capacity cDNA RT Kit
Reagents
Reagents:
- Applied Biosystems High Capacity cDNA Reverse Transcription Kit.
- Autoclaved 1.5 mL Eppendorf tubes.
- Thermal cycler
- Centrifuge with 96-well plate insert
- 96 well PCR plate and adhesive film
- Nuclease free dH20
2x RT master mix:
Keep all reagents on ice, including mastermix.
- Allow reagents to thaw on ice.
- For each sample (+ two controls), mix the follow:
- 2 μL 10✕ RT Buffer
- 0.8 μL 25✕ dNTP Mix
- 2.0 μL 10✕ RT Random Primers
- 1.0 μL RNase Inhibitor
- 3.2 μL Nuclease-free dH2O
- 1.0 μL MultiScribe Reverse Transcriptase
Remember: Do not add this to the RT-negative control. Use 1.0 μL dH20 instead.
- Mix gently by pipeting.
Protocol
Mix:
- Pipette 10 μL of 2✕ RT master mix into each well of a 96-well reaction plate or individual tubes.
- Pipette appropriate μL of RNA sample into each well (aiming for around 1000 ng pr. sample)
Remember: Do not add mRNA to the mRNA-negative control. Use 1.0 μL dH20 instead. - Top up to a total of 10 μL with nuclease-free dH20. Pipet up and down to mix. Total sample size should now be 20 μL.
- Seal the plates or tubes.
- Centrifuge the plate at 500 RPM for 1 minute to eliminate any air bubbles.
- Place the plate or tubes on ice until you are ready to load the thermal cycler.
Thermal cycler:
- Program thermal cycler as follows:
- Step 1: 25 °C for 10 min
- Step 2: 37 °C for 120 min
- Step 3: 85 °C for 5 min
- Step 4: 4 °C for ∞
- Load plate or tubes into cycler and run.
Storage:
- Transfer to 1.5 mL Eppendorf tube, label and store at -20 °C.
Quality control:
- ?
RETROscript Reverse Transcription Kit
Reagents
- Life Technologies, RETROscript Reverse Transcription Kit
- Autoclaved 1.5 mL Eppendorf tubes.
- PCR strips or plate with adhesive film.
- Thermal cycler
- Nuclease free dH20
Protocol
Preparation:
- Calculate amount of RNA to add. Aim for 1-2 μg total.
Note: Up to 5 ug can be used if target is tricky to find in down-stream applications. - Create master mix with following pr. sample:
- 2 μL 10x RT buffer
- 4 μL dNTP mix
- 1 μL RNase Inhibitor
- 1 μL MMLV reverse transcriptase
- Keep on ice till used.
Denature step:
- Working on ice, mix the following in a 1.5 Eppendorf tube:
- 1-2 μg RNA
- 2 μL Oligo(dT) or Random Decamers
- Up to 12 μL nuclease free water.
- Mix by pipetting gently up and down.
- Transfer solution to PCR tube, being careful not to introduce bubbles.
Note: Keep tubes on ice. - Turn on thermal cycler with the following program:
- 85°C for 3 minutes
- When plate reaches 85°C, pause, and place PCR tubes. Unpause program.
- Remove tubes from thermal cycler and place on ice.
Reverse transcription step:
- Add 8 μL of master max to each sample and mix by pipetting gently.
- Briefly spin down.
- Place back in thermal cycler, running the following program:
- 44°C for 1 hour
- 92°C for 10 minutes (to inactivate RT)
- 4°C hold forever
Storage and quality control:
- Transfer solution to labeled 1.5 Eppendorf tubes and store in -20°C or proceed to PCR.
References
- Applied Biosystems High Capacity cDNA Reverse Transcription Kit Protocol, http://docs.appliedbiosystems.com/pebiodocs/04375575.pdf
- Life Technologies RETROscript cDNA RT Kit protocol, https://tools.lifetechnologies.com/content/sfs/manuals/cms_056140.pdf
- cDNA Synthesis Protocol from Toni Vidal-Puig's lab: