Rapid Amplification of cDNA Ends

1. Experiment set up for 3’ RACE

1. Design a 5’ specific primer for a gene of interest (gene specific primer 1, GSP-1). It must be highly specific, as it is the only primer that introduces specificity. Therefore, a relatively long primer would be preferable (typical length around 24 nucleotides, Tm ranging from 55-65˚C). 
2. Design a second, nested primer (GSP-2), i.e. the primer should be located 3’ of GSP-1. This step is optional, but performing a second PCR with a nested primer will increase the yield and specificity of the PCR for the gene of interest. The primers used to amplify dSmad2 cDNAs are listed in Table 2.

2. Synthesis of cDNA 

1. Extract RNA from samples, using an RNA extraction method or kit following the manufacturer’s protocol. Treat the samples with DNase to avoid amplification of genomic DNA. In our example, we extracted RNA from 10 whole adult flies.
2. Measure the RNA concentration using a microvolume spectrophotometer and adjust with RNase free water to a maximum of 100 ng/μl if needed.
3. Prepare cDNA in a reverse transcription (RT) reaction, using a kit recommended for RACE. Prepare a master mix, with the following reagents per sample:
   - 4   μl  Reverse transcription buffer (5x)
   - 2   μl  Oligo(dT)₂₀ primer (an oligo consisting of 20 deoxythymidines)
   - 10 μl  RNA (max. 1 μg)
   - 3   μl  ddH₂O
   - 1   μl  reverse transcriptase 
   - TOTAL: 20 μl
4. Incubate the reaction for 90 min at 42ºC. Include a negative control omitting reverse transcriptase.
5. Inactivate the reverse transcriptase by incubating at 85ºC for 5 min.
6. In order to dilute DNA levels for efficient PCR, add 80 μl TE buffer to bring the total volume to 100 μl.

3. Amplification of cDNA fragments

1. Prepare the first-round amplification PCR mix by using a high-fidelity (HF) Taq polymerase:
   - 4      μl  5x HF Taq polymerase buffer
   - 0.4   μl  dNTP mix (10 mM each)
   - 1      μl  GSP1 primer (10 μM)
   - 1      μl  Oligo(dT)20 primer (10 μM)
   - 1      μl  cDNA template
   - 0.2   μl  HF Taq DNA polymerase
   - 12.4 μl  ddH2O
   - Total: 20 μl
2. Run the PCR by using the PCR1 program (Table 1). Include a negative control using as template the original RNA product without RT. Further, a ‘no primer’ control may be included.
3. Optional: nested PCR to increase specificity and the amount of cDNA products. Dilute 1 μl of the PCR products and negative control 1:20 in TE buffer. Use them as template for the second PCR reactions:
   - 4      μl  5x HF Taq polymerase buffer
   - 0.4   μl  dNTP mix (10 mM each)
   - 1      μl  GSP2 primer (10 μM)
   - 1      μl  Oligo(dT)₂₀ primer (10 μM)
   - 1      μl  PCR product template
   - 0.2   μl  HF Taq DNA polymerase
   - 12.4 μl  ddH2O
   - Total: 20 μl
4. Run the second PCR using the PCR2 program (Table 1)

4. Isolation of cDNA fragments

1. Prepare a 1-2% agarose gel with TAE buffer, using ethidium bromide or an alternative DNA stain. 
2. Mix 5 μl of sample with 1 μl of 6x gel loading buffer.
3. Load the samples and 1kb DNA ladder as marker and run the gel at 120 V for about 45 min or until the dye front is ~75% of the way down the gel. Ensure the samples do not run out of the gel.
4. Check the gel bands under a UV lamp. For further processing of the products, use low intensity UV, locate the amplified bands and cut them from the gel using a scalpel.
5. Purify the DNA from the gel by using a method like freeze-squeeze or a kit.
6. Store the purified cDNA fragments at -20ºC or use them immediately for further applications, such as sequencing or cloning.