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:
  1. One negative consisting of a duplicate sample but without the RT enzyme added.
  2. 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.

1. Allow reagents to thaw on ice.
2. 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.
3. Mix gently by pipeting.

Protocol

Mix:

1. Pipette 10 μL of 2✕ RT master mix into each well of a 96-well reaction plate or individual tubes.
2. 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.
3. 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.
4. Seal the plates or tubes.
5. Centrifuge the plate at 500 RPM for 1 minute to eliminate any air bubbles.
6. Place the plate or tubes on ice until you are ready to load the thermal cycler.

Thermal cycler:

1. 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 ∞
2. Load plate or tubes into cycler and run.

Storage:

1. Transfer to 1.5 mL Eppendorf tube, label and store at -20 °C.

Quality control:

1. ?

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:

1. 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.
2. 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:

1. 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.
2. Mix by pipetting gently up and down.

1. Transfer solution to PCR tube, being careful not to introduce bubbles.
   Note: Keep tubes on ice.
2. 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.
3. Remove tubes from thermal cycler and place on ice.

Reverse transcription step:

1. Add 8 μL of master max to each sample and mix by pipetting gently.
2. Briefly spin down.
3. 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:

1. 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: