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Rapid Amplification of cDNA Ends
 

Rapid Amplification of cDNA Ends

Article

Overview

Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Brückner1,2,3
1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research
2Department of Cell and Tissue Biology, 
3Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA    

Rapid Amplification of cDNA Ends (RACE) is a technique that allows amplification of full-length cDNA from mRNA by extending to the 3’ or 5’ end, even without prior knowledge of the sequence (Frohman et al., 1988). In contrast to regular PCR, it uses only one specific PCR primer and a second non-specific primer that will indiscriminately bind to most mRNAs. Depending on whether the area to be amplified is on the 3’ or 5’ end of the mRNA, the second primer is chosen to bind the polyA tail (3’ end) or a synthetic linker added to all transcripts (5’ end). These primer combinations are known to yield “one-sided” or “anchored” PCR due to PCR amplification using the known sequence of one side- 3’ or 5’(Ohara et al., 1989). The approach allows the capture of gene-specific rare mRNAs that otherwise would be hard to detect, e.g. because of their relatively low expression or unknown complete sequence (Frohman et al., 1988).

RACE is initiated with a reverse transcription step (RT) to synthetize single stranded complimentary DNA (cDNA) from mRNA. This is followed by two consecutive PCRs that amplify the cDNA fragments from a gene of interest. To perform RACE, the sequence of the gene of interest must be at least partially known, as it is needed to design the gene-specific primers (GSPs; Frohman, 1994; Liu and Gorovsky, 1993). As the second primer pair is a generic primer that will anneal to all transcripts present in the sample, the specificity of the PCR reactions is reduced. RACE is therefore ideally performed with two nested PCRs to lower the chances of amplifying non-specific transcripts (Figure 1). Depending on the direction of amplification relative to the GSP, the technique is categorized as 5’ or 3’ RACE (Figure 1). 

3’ RACE (Figure 1A) takes advantage of the poly(A) tail present in mRNAs as a generic binding site for the non-specific primer. This simplifies the technique, as one can synthetize cDNA by using oligo (dT) primers. GSPs are located in the 5’ region of the transcripts. This RACE variant allows the detection of transcript variants and different 3’ UTRs (Scotto Lavino et al., 2007a). In 5’ RACE (Figure 1B), GSPs are located in the 3’ region of the gene. To be able to use a non-specific primer that binds to all transcripts, an adapter that serves as generic primer binding site is attached to the 5’ RNA ends. To attach the adapter to the mRNA, the 5’ cap that protects mRNAs against exonucleases and promotes translation must be removed (Bird et al., 2016). Opposed to 3’ RACE, 5’ RACE helps finding differential 5’ splice variants and alternative 5’ UTRs (Scotto Lavino et al., 2007b).

Here we will perform 3’ RACE to identify and isolate different transcript variants using the known 5’ sequence (Figure 2A) encoded by the Drosophila dSmad2 (Smox) gene. dSmad2, a fly Smad protein, is an important transducer of Activin-β signaling, a pathway of the TGF-β superfamily. dSmad2 regulates multiple cellular processes, such as cell proliferation, apoptosis and differentiation (Upadhyay et al., 2017). As differentially spliced transcripts of dSmad2 may have different functions in the adult fly, a first step in exploring these potential functions is to assess all transcript variants of dSmad2 at this developmental stage.

Procedure

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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.

Typically, with novel mRNAs only a portion of its complete sequence is known. The missing nucleotide sequences at the ends of the mRNA can be determined using a PCR-based method called Rapid Amplification of cDNA Ends, or RACE.

In eukaryotes, mature mRNAs have distinctive structural features at both ends. At the five-prime end, most have a methylated guanosine residue connected to the mRNA via a five-prime to five-prime triphosphate linkage. This is also known as the five-prime cap. At the three-prime end, most eukaryotes have a tail of 20 to 250 adenylate residues, called the poly(A) tail.

Now, if the nucleotide sequence of even a small segment is known anywhere within the mRNA, the sequence up to its three-prime end can be amplified using a gene-specific primer and a non-specific oligo(dT) primer that anneals to the poly(A) tail at the three-prime end. This subset of the RACE technique is called three-prime RACE, and it allows for the detection of transcript variants and different three-prime Untranslated Regions, or UTRs.

The sequence at the five-prime end can be amplified similarly. To do this, a poly(A) tail is first attached at the five-prime end. Then, using a non-specific oligo(dT) primer that anneals to the appended tail and a gene-specific primer, the sequence up to the five-prime end can be amplified. This subset of RACE is known as five-prime RACE and is used to find differential five-prime splice variants and alternative five-prime UTRs.

To perform three-prime RACE to identify different transcripts encoded by a given gene, RNA is first isolated from the organism or tissue of interest. Next, cDNA is synthesized from the isolated mRNAs with a reverse transcription reaction that utilizes an oligo(dT) primer and a reverse transcriptase enzyme, which generates complementary DNA from an RNA template. Next, from the generated pool of cDNAs, the target cDNA's unknown three-prime end is extended via PCR, utilizing the non-specific oligo(dT) primer and a gene-specific primer, and amplified during the PCR reaction.

However, due to the generic nature of the non-specific primer and random mispriming by the gene-specific primer, they can anneal to off-target cDNAs, causing them to amplify as well. To overcome this problem, a second round of PCR is conducted using nested primers, which bind downstream of the first set of primers. This second set of primers further amplifies the cDNA of interest but not the non-specific product, increasing the specificity and yield of the reaction.

Finally, the PCR products are separated using agarose gel electrophoresis, which separates different transcripts of the target gene based on their sizes, producing distinct bands. The band of interest can then be excised, purified, and finally sequenced to obtain the transcript's complete sequence.

In this video, we will demonstrate the three-prime RACE technique to identify and isolate different transcripts encoded by the Drosophila dSmad2 gene.

Before beginning the synthesis and amplification of the cDNA, use a primer design software to create a five-prime specific primer for the gene of interest, dSmad2 in this example. To ensure the primer is highly specific, it should be around 24 nucleotides and have a Tm ranging from 55 to 65 degrees Celsius. Next, design a second nested primer located three-prime of the first primer, which is also specific for the target sequence.

To begin, extract RNA from 10 whole adult flies with a commercially available RNA extraction kit. Once the RNA is extracted, resuspend the pellet in 20 microliters of nuclease-free water. For a 50-microliter reaction, add 5 microliters of the reaction buffer to each sample tube, and bring up the volume of the reaction by adding 24 microliters of nuclease-free water. Then, add 1 microliter of DNase I to prevent amplification of genomic DNA. Finally, incubate the samples at 37 degrees Celsius for 15 minutes.

After DNase treatment, inactivate the enzyme with 1 microliter of 25 millimolar EDTA in each of the tubes. Add the sample to a spin column to purify the RNA. Next, use a microvolume spectrophotometer to measure the RNA concentration. Adjust the concentration to 100 nanograms per microliter by adding nuclease-free water.

Now, prepare a master mix for the reverse transcription reaction. In addition to the test DNA samples, include one negative control by omitting reverse transcriptase from the appropriate tube. Incubate the reactions for 90 minutes at 42 degrees Celsius. Then, inactivate the reverse transcriptase by incubating the tubes at 85 degrees Celsius for five minutes. Next, dilute the DNA levels by adding 80 microliters of TE buffer to each tube to bring the reaction total to 100 microliters.

Now that the cDNA is synthesized, amplify the gene of interest via PCR. To do this, first prepare the first round amplification PCR mix. In addition to the cDNA template, include a negative control reaction that uses the template from the non-reverse-transcribed product, as well as a reaction that does not have the gene-specific primer as a no-primer control.

Once the reactions are prepared, carry out PCR amplifications in a thermocycler equipped with a heated lid. Next, dilute the products 1 to 20 by adding 1 microliter of the PCR products to 19 microliters of TE buffer. Using these diluted products, prepare the second round amplification PCR mix. Then, use a thermocycler to run the second PCR.

To isolate the PCR fragments, prepare a 1% agarose gel by adding 1 gram of agarose per 100 milliliters of TAE buffer. Melt the mixture for two minutes in the microwave, and then add SYBR Safe stain or ethidium bromide. Pour the molten mixture in a gel tray and allow it to set.

While the gel is setting, pipette 1 microliter droplets of 6X Loading Dye onto a piece of parafilm corresponding to the number of samples. Add 5 microliters of sample to each droplet. Now, load the samples, along with a 1 kilobase DNA ladder, onto the gel. Run the gel at 120 volts for approximately 45 minutes or until the dye front is 75% of the way down the gel.

When the gel has finished running, check the gel bands under a UV transilluminator. Locate the bands and cut them out using a scalpel. Purify the cDNA fragments using a commercially available spin column kit. Once purified, the cDNA fragments can be stored at minus 20 degrees Celsius or used immediately for further analysis.

Prior to this experiment, two transcripts for Drosophila dSmad2 to were annotated in the Drosophila genomic database, FlyBase. Based on the expected splicing of this gene, two products should be identified by the three-prime RACE protocol.

The results from this experiment, however, reveal three different transcripts for dSmad2. Among the predicted products, one transcript is predominant, and one is expressed at a lower level. In addition, a previously undescribed smaller product, visible at 750 base pairs, was detected.

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Results

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There are two annotated transcripts for Drosophila dSmad2 in FlyBase (Fig. 2A). Our results, however, reveal three different transcripts for dSmad2, ranging in size from 750 bp to 1400 bp (Fig. 2B). Differences in the intensity of the bands indicate their relative expression levels. Among the predicted products, one transcript is predominant (Fig. 2B, black arrow A), and one is expressed at a lower level (Fig. 2B, black arrow B). In addition, a previously undescribed smaller product was detected (Fig. 2B, grey arrow C).

The identification of such rare transcripts is only possible with a sensitive method such as RACE. Following RACE, one can clone the fragments obtained by PCR to study the transcript sequence, find its similarity with other transcript variants and clone it for transgenesis. Such experiments help investigate the functions of transcript variants specific to a tissue or developmental stage.

Figure 1
Figure 1. Schematics of the two different RACE approaches. A) 3’ RACE. cDNA is synthesized by using oligo (dT) primers. The same oligo (dT) primer is used in combination with a 5’ gene-specific primer to amplify 3’ cDNA ends through one, or better two, rounds of PCR. B) 5’ RACE. RNA is first decapped to free the 5’ end. In a second step, an adapter RNA sequence is added to the 5’ end. cDNA is generated by using a primer complementary to the adapter sequence. The same primer is used in combination with 3’ gene-specific primer/s to amplify the cDNA. 

Figure 2
Figure 2. A) RACE allows amplification of several transcripts from the same gene, exemplified by 3’ RACE of Drosophila dSmad2. Combination of a nonspecific primer (Oligo dT) with a gene-specific primer (GSP) yields cDNAs of different lengths (Product A, Product B) corresponding to alternatively spliced transcripts (mRNA A, mRNA B). B) Separation of PCR products of dSmad2 3’ RACE on a 1% agarose gel. Lanes correspond to (1) 1 kb DNA ladder as marker, (2) no RT negative control (contains RNA and primers), (3) no primers negative control (contains cDNA template) (4) full reaction of 3’ RACE for dSmad2 (contains. primers and cDNA template). Amplification of dSmad2 transcripts by 3’ RACE yields three distinct products (arrows). Two cDNAs were of predicted sizes around 1400 and 1200bp (black arrows A and B, corresponding to Product A and B in (A)). In addition, a previously undescribed smaller cDNA of ~750bp was detected (grey arrow, C). 

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Applications and Summary

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RACE provides a quick, inexpensive and powerful tool to obtain cDNAs from sequences that are only partially known, or from rare transcripts that are otherwise harder to amplify. It can be used to either find the sequence of unknown transcripts from an already known gene or to clone such transcripts for further studies. Following RACE, such transcripts can be overexpressed in cell-based systems or model organisms to investigate their function in vivo.

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Acknowledgments

This work was supported by grants from the National Science Foundation # IOS-1355222 and the National Institutes of Health # 1R01GM112083 and 1R01GM131094 (to K.B.).

References

  1. Bird, J.G., Zhang, Y., Tian, Y., Panova, N., Barvík, I., Greene, L., Liu, M., Buckley, B., Krásný, L., Lee, J.K., et al. (2016). The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA. Nature 535, 444–447.
  2. Frohman, M.A. (1994). On beyond classic RACE (rapid amplification of cDNA ends). PCR Methods Appl. 4, S40–S58.
  3. Frohman, M.A., Dush, M.K., and Martin, G.R. (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. U.S.a. 85, 8998–9002.
  4. Liu, X., and Gorovsky, M.A. (1993).  Mapping the 5′ and 3′ ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE). Nucleic Acids Res. 21, 4954–4960.
  5. Ohara, O., Dorit, R.L., and Gilbert, W. (1989). One-sided polymerase chain reaction: the amplification of cDNA. Proc. Natl. Acad. Sci. U.S.A. 86, 5673–5677.
  6. Scotto Lavino, E., Du, G., and Frohman, M.A. (2007a). 3′ End cDNA amplification using classic RACE. Nat Protoc 1, 2742–2745.
  7. Scotto Lavino, E., Du, G., and Frohman, M.A. (2007b). 5′ end cDNA amplification using classic RACE. Nat Protoc 1, 2555–2562.
  8. Upadhyay, A., Moss-Taylor, L., Kim, M.-J., Ghosh, A.C., and O’Connor, M.B. (2017). TGF-β Family Signaling in Drosophila. Cold Spring Harb Perspect Biol 9, a022152.

Transcript

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