3´ RACE System for Rapid Amplification of cDNA Ends

Principles of RACE

Rapid Amplification of cDNA Ends (RACE) is a procedure for amplification of nucleic acid sequences from a messenger RNA template between a defined internal site and either the 3´ or the 5´ end of the mRNA (1). This methodology of amplification with single-sided specificity has been described as “one-sided” PCR (2) or “anchored” PCR (3). PCR requires two sequence-specific primers that flank the sequence to be amplified (4,5). However, to amplify and characterize regions of unknown sequences, this requirement imposes a limitation (3). 3´ RACE takes advantage of the natural poly(A) tail found in mRNA as a generic priming site for PCR. In this procedure, mRNAs are converted into cDNA using reverse transcriptase (RT) and an oligo-dT adapter primer. Specific cDNA is then amplified by PCR using a gene-specific primer (GSP) that anneals to a region of known exon sequences and an adapter primer that targets the poly(A) tail region. This permits the capture of unknown 3´-mRNA sequences that lie between the exon and the poly(A) tail. 5´ RACE uses an antisense gene specific primer for the synthesis of specific cDNA by reverse transcriptase.

Prior to PCR, a TdT-tailing step attaches an adapter sequence to the unknown 5´ sequences of the cDNA. Specific cDNA is then amplified by PCR using a GSP that anneals in a region of known exon sequences and an adapter primer that targets the 5´ terminus. RACE has been used for amplification and cloning of rare mRNAs (6) and may be applied to existing cDNA libraries (7). Additionally, RACE products can be directly sequenced without any intermediate cloning steps (8,9), or the products may be used to prepare probes (10). Products generated by the 3´ and 5´ RACE  procedures may be combined to generate full-length cDNAs (10,11). Lastly, the RACE procedures may be utilized in conjunction with exon-trapping methods (12) to enable amplification and subsequent characterization of unknown coding sequences.

Summary of the 3´ RACE System

The 3´ RACE procedure is summarized as follows. First strand cDNA synthesis is initiated at the poly(A) tail of mRNA using the adapter primer (AP). After first strand cDNA synthesis, the original mRNA template is destroyed with RNase H, which is specific for RNA:DNA heteroduplex molecules. Amplification is performed, without intermediate organic extractions or ethanol precipitations, using two primers: one is a user-designed GSP that anneals to a site located within the cDNA molecule; the other is a universal amplification primer that targets the mRNA of the cDNA complementary to the 3´ end of the mRNA. Two universal amplification primers are provided with the system. The universal amplification primer (UAP) is designed for the rapid and efficient cloning of RACE products using the uracil DNA glycosylase (UDG) cloning method (13–16). The abridged universal amplification primer (AUAP) is homologous to the adapter sequence used to prime first strand cDNA synthesis.

Since the 3´ RACE System utilizes the poly(A) tail region as an initial priming site, multiple amplification products  may be synthesized, depending on the degree of specificity conferred by the GSP. To generate a specific amplification product, the user may find it advantageous to design a second “nested” GSP, as recommended by Frohman et al. (10) and reamplify the RACE products; this procedure is discussed in greater detail at the end of this chapter

Isolation of Total RNA

One of the most important factors preceding the synthesis of substantially full-length cDNA is the isolation of intact RNA. The quality of the RNA dictates the maximum amount of sequence information that can be converted into cDNA. Thus, it is important to optimize the isolation of RNA from a given biological source and to prevent adventitious introduction of RNases (17) and inhibitors of reverse transcriptase such as guanidinium salts, SDS and EDTA (18). RNA can be isolated using a variety of methods. The recommended method for 3´ RACE is the guanidine isothiocyanate/acid-phenol method originally described by Chomzynski and Sacchi (19). The TRIzol Reagent method is an improvement of the original single-step method of Chomczynski and Sacchi (20) and can be used for the preparation of RNA from as little as 10³ cells or milligram quantities of tissue (21). Total RNA isolated with Invitrogen TRIzol Reagent is undegraded and essentially free of protein and DNA contamination. For the isolation of RNA from small quantities of sample (<10⁶ cells or <10 mg tissue) without using phenol, the GlassMAX RNA Microisolation Spin Cartridge System is recommended (22).

Total RNA isolated by these methods may contain small amounts of genomic DNA that may subsequently be amplified along with the target cDNA. The presence of this DNA is not likely to cause problems because it lacks the poly(A) region present in the mRNA analyte. As a precaution, however, we recommend performing a control experiment without reverse transcriptase to determine whether a given fragment is of genomic DNA or of cDNA origin. Products generated in the absence of RT are of genomic origin. If your application requires removal of all genomic DNA from your RNA preparation, refer to DNase I Digestion of RNA Preparation.

First Strand cDNA Synthesis from Total RNA

The first strand cDNA synthesis reaction is catalyzed by Invitrogen SuperScript II RT. This enzyme is a mutant of M-MLV RT that has been engineered to reduce RNase H activity, resulting in greater yields and more full-length synthesis (23,24,25). The enzyme exhibits increased thermal stability and may be used at temperatures up to 50°C. In addition, SuperScript II RT is not inhibited significantly by ribosomal and transfer RNA and may be used to synthesize first strand cDNA from a total RNA preparation. The RNA template is removed from the cDNA:RNA hybrid molecule by digestion with RNase H after cDNA synthesis to increase the sensitivity of PCR (26). The AP which primes first strand cDNA synthesis, has been engineered to contain three restriction endonuclease sites and a Not I half-site. Inclusion of these sequences in the primer may facilitate post-amplification cloning using either a restriction endonuclease-based (27) or a T4 DNA polymerase-based (28) method. Because the AP initiates cDNA synthesis at the poly(A) region of the mRNA, it effectively selects for polyadenylated mRNAs; thus, oligo(dT)-selection for poly(A)+ RNA is typically not necessary although incorporating this step may facilitate the detection of rare mRNA transcripts.

Amplification of a Target cDNA

Amplification of a target cDNA requires priming with two oligonucleotides and Taq DNA polymerase. The sense amplification primer is the user-provided GSP,which is specific for the particular gene or sequence of interest and may be designed to include sequence elements that facilitate subsequent cloning steps. The antisense amplification primer is one of the two universal amplification primers provided with the system. The AUAP contains a restriction endonuclease site sequence (adapter region) homologous to the adapter region of the AP. The UAP is composed of the same adapter region plus a dUMP-containing sequence at the 5´ end of the primer required for UDG-mediated cloning. The UAP should not be used to prime DNA synthesis with any archaeobacterial polymerase (e.g., Pyrococcus furiosus, Pyrococcus woesei, etc.) or any long PCR enzyme mixture (e.g., Elongase® Enzyme Mix) that contains one of these enzymes due to the inhibition of polymerase activity by dUMP-containing DNA. Both the AUAP and the UAP will function in PCR at annealing temperatures up to 68°C.e

Design of the Gene-Specific Primer

Efficient and specific PCR amplification is highly dependent on primer design. This is especially true for RACE applications since the PCR is carried out with only a single GSP. In general, effective primers form stable duplexes with their target sequences, are highly specific for their target sequences, and are free of secondary structure such as hairpin loops and dimers (29–31). Additionally, the complementarity of primer 3´-termini must be minimized since primer-dimer artifacts may significantly reduce PCR efficiency. Therefore, dimer formation with the AUAP or UAP primer, as well as itself, should be reduced. Computer algorithms that have been developed (32–35) and are commercially available often facilitate this analysis. Discussion of primer design for RACE applications may be found in Frohman (11) and Loh (6). It should be noted that in cases where only limited peptide sequence information is available, a degenerate GSP may be prepared. The AUAP and UAP included wit  the system have been engineered to function at PCR annealing temperatures up to 68°C and to facilitate the cloning step. The user-defined GSPs need to be compatible with the cloning method. Add the following to the 5’ end of the GSP: for UDG cloning: 5´–CAU CAU CAU CAU–3´ (use with UAP) for T4 DNA polymerase cloning: 5´–CGA–3´ (use with AUAP)

Nested Amplification

The AP is designed to synthesize first strand cDNA from all polyadenylated mRNAs. The sequence specificity in the amplification reaction is therefore derived solely from the GSP. Often, a second “nested” GSP may be utilized in conjunction with the AUAP or UAP in a second amplification reaction to give the 3´ RACE procedure the specificity of a second primer (9). The nested GSP can anneal immediately adjacent to the first GSP or at sequences within the cDNA further downstream. The nested amplification reaction may be conveniently conducted using a plug of agarose from the gel analysis of the initial 3´ RACE reaction (see Nested Amplification from an Agarose Plug). Ultimately, the 3´ RACE procedure should produce a single, prominent band on an agarose gel. When performing 3´ RACE with a nested primer, sequences specific for subsequent cloning manipulations (see Design of the Gene-Specific Primer) must be designed into the nested GSP.


Cloning of Amplification Products

Conventional cloning methods that typically involve end-repair and blunt-end cloning can be problematic for amplified products (36–38). An alternative is a rapid and efficient method involving the use of UDG (13–16). This method requires that the user design a GSP containing containing a 5'-(CAU)4 sequence. Incorporation of dUMP into the GSP may be accomplished on most automated synthesizers or with Invitrogen Custom Primers (see Design of the Gene-Specific Primer). The product of the 3´ RACE reaction primed with the UAP and the dUMP-containing GSP is treated with UDG, which converts dUMP residues to abasic sites (39,40), to generate 3´ overhangs. The directional nature of the UDG cloning process can be exploited to lend an added level of specificity to the RACE procedure. Only amplification product that results from priming by both the UAP and the appropriately designed GSP are efficient substrates for UDG cloning. Another alternative to conventional cloning methods uses the 3´ to 5´ exonuclease activity of T4 DNA polymerase as the basis for cloning as described by Stoker (28). In this procedure, the AUAP is used in the amplification reaction, and the 3´ RACE products are treated with T4 DNA polymerase to generate a Not I 5´ overhang. Similarly, the user may design a site into the GSP (see  Design of the Gene-Specific Primer). Another approach to cloning is to digest the 3´ RACE product using one of the restriction endonuclease sites designed into the AUAP.  The user may also design unique restriction sites into the GSP, exploit a site present in the cDNA sequence or end-repair the 3´ RACE product prior to restriction endonuclease digestion (37).

Components

Components are provided in sufficient quantities to perform 20 separate reactions, each converting 1-5 μg of total RNA into first strand cDNA. A control RNA and amplification primers are included in the system to verify performance of the first strand cDNA synthesis reaction and subsequent amplification.  Note: The 3´ RACE System does not include Taq DNA polymerase or the reagents required for cloning. Store the 3´ RACE System at –20°C.

Advance Preparations

Please review the protocols before using this system. You will need the following items not included in the system:

• sterilized 0.5-ml microcentrifuge tubes;
• automatic pipets capable of dispensing 1 to 20 μl and 20 to 200 μl;
• sterilized, RNase-free disposable tips for automatic pipets;
• disposable latex gloves;
• sterilized, distilled water;
• GSP (user-defined, appropriately engineered);
• microcentrifuge capable of generating a relative centrifugal force of 14,000 × g;
• 37°C, 42°C, and 70°C water baths or heat blocks;
• Taq DNA polymerase;
• chloroform;
• mineral oil; and
• thin-walled PCR tubes.

Protocol 1. First Strand cDNA Synthesis

This procedure converts 1 to 5 μg of total RNA into first strand cDNA. Poly(A)+ RNA may be used in this protocol, but is typically not necessary. A control RNA is included in the 3´ RACE System as an aid in verifying that the system performs in your hands. If you decide to use the control RNA as a template for first strand synthesis, simply substitute 2 μl of control RNA (100 ng) in the first strand reaction for your total RNA.

1. Mix and quickly centrifuge each component before use.

Note:


2. Combine 1-5 μg of total RNA or 50 ng of poly(A)+ RNA and DEPC-treated water to a final volume of 11 μl in a 0.5-ml microcentrifuge tube.


3. Add 1 μl of the 10 μM AP solution, mix gently, and collect reaction by brief centrifugation.
   
   Note:   If you have >5 μg of total RNA, increase reaction volumes and amount of SuperScript II RT proportionately. If you have <1 μg of total RNA, no changes to the protocol are necessary. 50 to 500 ng of poly(A)+ RNA may be substituted for total RNA in this protocol
   
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4. Heat the mixture to 70°C for 10 min and chill on ice for at least 1 min. Collect the contents of the tube by brief centrifugation and add the following:
   
   
   

   Component	Volume (μl)
   10X PCR buffer	2
   25 mM MgCl2	2
   10 mM dNTP mix	1
   0.1 M DTT	2

   
   Final composition of the reaction:
   
   20 mM Tris-HCI (pH 8.4 at 22°C)
   50 mM KCI
   2.5 mM MgCI₂
   10 mM DTT
   500 nM AP
   500 μM each dATP, dCTP, dGTP, dTTP
   1-5 μg (≤50 ng/μl) of RNA


5. Mix gently and collect the reaction by brief centrifugation. Equilibrate the mixture to 42°C for 2 to 5 min.


6. Add 1 μl of SuperScript II RT. Incubate the tube in a 42°C water bath or heat block for 50 min.


7. Terminate the reaction by incubating at 70°C for 15 min.


8. Chill on ice. Collect the reaction by brief centrifugation. Add 1 μl of RNase H to the tube, mix, and incubate for 20 min at 37°C before proceeding toProtocol 2.
   
   


9. The reaction mixture may be stored at –20°C.
   
   Note:   You may stop at the end of step 9.
   
   Protocol 2. Amplification of the Target cDNA
   
   Optimal conditions for amplification are dependent on the nature of each particular primer and target sequence used. Alteration of the magnesium ion, dNTP, or primer concentration, as well as the thermocycling protocol, may be required. The optimal free magnesium concentration for efficient amplification is reported to be between 0.7 to 0.8 mM (29). Since magnesium binds deoxyribonucleoside triphosphates, this factor is affected by both primer and dNTP concentration. In general, lower concentrations of dNTP (50 to 200 μM), MgCl₂ (1 to 1.5 mM), and primer (0.1 to 0.2 μM) promote higher fidelity and specificity (41). Higher nucleotide concentration, however, can be used to improve product yield as well as to promote 3´-terminal T-mismatches (42). For a detailed discussion of parameters affecting PCR, please refer to Innis and Gelfand (43) or Saiki (29,30).
   
   The addition of either Taq DNA polymerase, dNTPs, or MgCl₂ after reactions have been equilibrated at 75°C to 80°C has been reported to improve the specificity of the reaction (44,45). This “hot start” practice reduces nonspecific binding and the extension of primers during the initial denaturation process. A practical alternative to this classic “hot start” method is to set up reactions on ice then place complete PCR mixtures in a thermal cycler equilibrated to 80°C to 90°C.
   
   
   1. To a fresh 0.5-ml microcentrifuge tube, add the following:
   
   

   Component	Volume (μl)
   10X PCR buffer	5
   25 mM MgCl2	3
   autoclaved, distilled water	36.5
   10 mM dNTP mix	1
   GSP (prepared as 10 μM solution)	1
   AUAP (10 μM) or UAP (10 μM)	1
   Taq DNA polymerase (2 to 5 units/μl)	0.5

   
   Note:  
   
   
   
   
   
   ₂
   
   
   
   
   
   
   2. Add 2 μl from the cDNA synthesis reaction to the tube. Mix gently and layer 75 μl of mineral oil over the reaction. Collect the reaction briefly by centrifugation.
      
      Note:   Mineral oil is necessary only when using thermal cyclers that require this barrier.
   
   
   3. Incubate the reaction at 94°C for 3 min. (Extended preamplification denaturation times may impair the efficiency of long (>2 kb) target sequences (46).
   
   
   4. Perform 20 to 35 cycles of PCR, using the protocol accompanying Taq DNA polymerase.
      
      Note:  Step 5 is unnecessary if no mineral oil was used in step 2.
   
   
   5. Following amplification, extract the sample with 50 μl of chloroform. Transfer the aqueous layer to a fresh tube.
   
   
   6. Analyze 10 to 20 μl of the amplified sample, using agarose gel electrophoresis and ethidium bromide staining, and the appropriate molecular size standards. If the positive control RNA was used, a single 720-bp band will be visible (see Testing the 3´ RACE System).
      
      Note:   If sequences are available for use as internal probes, it is strongly recommended that Southern blot analysis be used to confirm the identity of specific product bands. Specific product can also be identified using a diagnostic restriction endonuclease digestion if the amplified cDNA sequence contains a known restriction site.
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General Suggestions

The control RNA provided with the 3´ RACE System can be used to troubleshoot both the first strand reaction and the amplification reaction. Use the following protocols, as needed, to troubleshoot for particular problems. To perform th  3´ RACE procedure using the control RNA, you may need the following items, in addition to those listed in Advance Preparations.

• [α-³²P]dCTP (3,000 Ci/mmol; 10 mCi/ml)
• glass-fiber filters
• heat lamp
• 10% (w/v) TCA containing 1% (w/v) sodium pyrophosphate
• 95% ethanol

First Strand cDNA Synthesis

1. Label two autoclaved 0.5-ml microcentrifuge tubes “A” and “B.” Tube A will have an addition of radioisotope t  determine the efficiency of first strand synthesis. An aliquot from tube B will be used for amplification.
2. Prepare the RNA:primer mixture in a sterile 0.5-ml tube:


Volume (μl)

Component	Tube A	Tube B
control RNA (50 ng/μl)	2	2
AP (10 μM)	1	1
DEPC-treated water	8	9
final volume	11	12



3. Incubate at 70°C for 10 min and place on ice for 1 min. Collect the contents of each tube by brief centrifugation and add the following to each tube:



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Volume (μl)

Component	Tube A	Tube B
10X PCR buffer	2	2
25 mM MgCl2	2	2
10 mM dNTP mix	1	1
0.1 M DTT	2	2
SuperScript II RT (200 units/μl)	1	1



4. Add 1 μl of [α-³²P]dCTP (3,000 Ci/mmol; 10 mCi/ml) to tube A. Final volume will be 20 μl.


5. Mix gently and collect the reaction by brief centrifugation.


6. Incubate for 42°C for 50 min.


7. Terminate both reactions by incubating at 70°C for 15 min. Place on ice for 10 min.


8. Collect each reaction by brief centrifugation. Add 1 μl of RNase H to tube B and incubate for 20 min at 37°C. Proceed with tube B to Section Amplification of the Control-Synthesized cDNA.


9. Add 80 μl of distilled water to tube A and mix gently.


10. Remove two 5-μl aliquots from tube A and spot the aliquots onto glass-fiber filters. Dry one of the filters under a heat lamp or at room temperature. This filter will be used to determine the specific activity of the dCTP reaction.


11. Wash the other filter three times in sequence, for 5 min each time, with 50 ml of ice-cold, 10% (w/v) TCA containing 1% (w/v) sodium pyrophosphate. Wash the filter once with 50 ml of 95% ethanol at room temperature for 2 min. Dry the filter under a heat lamp or at room temperature. This filter will be used to determine the yield of first strand cDNA.


12. Count both filters in standard scintillant to determine the amount of 32P in the reaction, as well as the amount of 32P that was incorporated.


13. Using equation 1, determine the specific activity (SA) of the dCTP in the first strand reaction from the counts obtained from the unwashed filter:
    
                                                          cpm/5 μl
    SA (cpm/pmol dCTP) = __________________
                                                 500 pmol dCTP/5 μl


14. Using equation 2, determine the yield of cDNA from the counts obtained from the washed filter and the specific activity calculated from the unwashed filter:
    
                                                    (cpm) × (100 μl/5 μl) × (4 pmol dNTP/pmol dCTP)
    Amount of cDNA (μg) = ________________________________________
    
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                                                                (SA) × (3,030 pmol dNTP/μg cDNA)
    
    The yield calculated for the labeled cDNA in tube A can be assumed as equivalent to that of the unlabeled cDNA in tube B.


15. Following first strand cDNA synthesis from the control RNA, you may wish to analyze the remaining cDNA in tube A by alkaline agarose gel electrophoresis or denaturing polyacrylamide gel electrophoresis (PAGE).
    
    Amplification of the Control-Synthesized cDNA
    
    Following first strand cDNA synthesis from the control RNA, the control GSP  and the UAP can be used to troubleshoot the amplification reaction. Electrophoretic analysis of DNA products amplified using the control primers should yield a prominent 720-bp band.
    
    5´–CAU CAU CAU CAU GAC CGT TCA GCT GGA TAT TAC–3´
    
    
    1. In each of three fresh 0.5-ml microcentrifuge tubes, prepare a PCR mixture as in step 1 of Protocol 2, substituting 1 μl of the control GSP for the user-defined GSP and using the UAP amplification primer. Place tubes on ice.
    2.  In separate 1.5-ml microcentrifuge tubes, prepare serial dilutions of the control cDNA from tube B in sterile, distilled water. Dilute 1:100, 1:1,000, 1:10,000, 1:100,000, and 1:1,000,000. Use the three highest dilutions: control 1: 1:104 (~106 input RNA molecules/μl) control 2: 1:105 (~105 input RNA molecules/μl) control 3: 1:106 (~104 input RNA molecules/μl)
    3.  Add 2 μl from each control dilution to a separate reaction tube from step 1. Mix and layer 75 μl of mineral oil over each reaction. Collect each reaction by brief centrifugation.
    4.  Perform steps 3 through 6 in Protocol 2. Amplify with 30 cycles of 94°C for 45 s, 55°C for 25 s, and 72°C for 3 min. Following agarose gel electrophoresis and ethidium bromide staining, you should be able to visualize a 720-bp band for all three concentrations of input cDNA.
    
    Minimizing RNase Contamination
    
    Successful cDNA synthesis demands an RNase-free environment at all times, which will generally require the same level of care used to maintain aseptic conditions when working with microorganisms. Several additional guidelines should be followed:
    
    
    1. Never assume that anything is RNase-free. Use sterile pipettes, centrifuge tubes, culture tubes, or any similar labware that is explicitly stated to be sterile. Note:  Wear latex gloves for all manipulations involving RNA.
    2. Dedicate a separate set of automatic pipettes for manipulating RNA and the buffers and enzymes used to synthesize cDNA.
    3. Avoid using any recycled glassware unless it has been specifically rendered RNase-free by rinsing with 0.5 N NaOH followed by copious amounts of sterilized, distilled water. Alternatively, bake glassware at 150°C for 4 h.
    4. Microcentrifuge tubes can generally be taken from an unopened box, autoclaved, and used for all cDNA work. RNase-free microcentrifuge tubes can be purchased from several suppliers. If it is necessary to decontaminate untreated tubes, soak the tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPC), rinse with sterilized, distilled water, and autoclave.
    5. If made with RNase-free labware, most solutions can be made from reagent-grade materials and distilled water, and then autoclaved. Prepare heat-sensitive solutions using sterilized, distilled water, and filtering through a 0.2 μm using sterilized, disposable unit.
    6.  Most aqueous buffer solutions can be treated with 0.01% (v/v) DEPC and autoclaved. Note: Buffers containing primary amines (such as tris) cannot be effectively treated by this method.
    7.  Use aerosol-resistant pipette tips.
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