5′ RACE Protocol using the Template Switching RT Enzyme Mix (NEB #M0466)

Rapid amplification of cDNA ends (RACE) is a widely used technique to identify the 5′ (5′ RACE) or the 3′ (3′ RACE) end of an RNA transcript when its sequence is only partially known. This 5′ RACE protocol contains two steps. In the first step, template switching reverse transcription reaction generates cDNAs with a universal sequence of choice, introduced by a template switching oligo (TSO), attached to the 3′ end of the cDNA (5′ end of the transcript). In the second step, the 5′ end of the transcript can be identified via PCR amplification with primers that are specific to the gene of interest and the TSO handle.

Sample Recommendations:

To identify transcription start sites, high quality intact RNA is required, usually in the range of 10 ng - 1 μg. The RNA sample should be free of salts (e.g., Mg2+, guanidinium), divalent cation chelating agents (e.g., EDTA, EGTA, citrate) or organics (e.g., phenol, ethanol). If an excess amount of genomic DNA is present, an optional DNase I treatment can be performed. Inactivate or remove DNase I after treatment.

 

Reaction Preparation

    1. Briefly centrifuge the Template Switching RT Enzyme Mix to collect the solution to the bottom of tube, then place on ice.
    2. Thaw the Template Switching RT Buffer at room temperature completely. Vortex and centrifuge briefly to collect the solution to the bottom of tube, then place on ice.

cDNA Synthesis and Template Switching

Please note that the volume needed for each reagent is based on a final cDNA Synthesis and Template Switching reaction volume of 10 μl. If desired, the reaction can be scaled up proportionally, while observing the RNA input amount limit, to a final volume of 20 μl.

 

1. Primer Annealing for First Strand Synthesis

1.1 To anneal the RT primer with RNA templates, in a 0.2 ml nuclease free PCR tube, prepare the reaction as follows (on ice):

Reagent

Volume

Final Concentration

RNA

Up to 4 μl

10 ng – 1 ug

RT primer (10 μM)

1 μl

1 μM

dNTP (10 mM)

1 μl

1 mM

Nuclease-free Water

Variable

-

Total Volume

6 μl

-

Mix thoroughly by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube.

1.2 Incubate for 5 minutes at 70°C in a thermocycler with the lid temperature set at ≥85°C, then hold at 4°C until the next step.

 

2. Reverse Transcription (RT) and Template Switching

2.1 During the primer annealing reaction, vortex the Template Switching RT Buffer briefly followed by a quick spin to collect the solution to the bottom of the tube, then prepare the RT reaction mix as follows (adding RT Enzyme Mix last):

Reagent

Volume

Final Concentration

Template Switching RT Buffer (4X)

2.5 μl

1X

Template Switching Oligo (TSO) (75 μM)

0.5 μl

3.75 μM

RT Enzyme mix (10X)

1 μl

1X

Total Volume

4 μl

-

Mix thoroughly by gently pipetting up and down at least 10 times, then centrifuge briefly to collect solution to the bottom of the tube.

2.2 Combine 4 μl RT reaction mix (above) with 6 μl of the annealed mix from step 1.2, mix well by pipetting up and down gently at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube.

2.3 Incubate the 10 μl combined reaction in a thermocycler with the following steps:

90 minutes at 42°C
5 minutes at 85°C
Hold at 4°C

If not proceeding to the PCR Amplification step immediately, samples can be stored at 4°C overnight or at -20°C for up to a week.

 

PCR Amplification of 5’ Region of Transcripts

1. PCR Template Preparation

Dilute the RT reaction from step 2.3 above by 2-fold with water and use 1 μl of the diluted cDNA in a subsequent 25 μl PCR reaction. For low abundant RNA targets, up to 2.5 μl of undiluted cDNA can be used in a 25 μl PCR reaction.

2.PCR Reaction Set up

2.1 Assemble the PCR reaction on ice as follows:

Components

Volume

Final Concentration

Diluted template switching cDNA product

1 μl

-

Q5 Hot Start High-Fidelity Master Mix (2X) (NEB #M0494)

12.5 μl

1X

TSO-specific primer (10 μM)

1.25 μl

0.5 μM

Gene-specific reverse primer (10 μM)

1.25 μl

0.5 μM

H2O

9 μl

-

Total Volume

25 μl

-

2.2 Mix gently by pipetting up and down at least 10 times, then centrifuge briefly to collect solution to the bottom of the tube.

2.3 Incubate the reaction in a thermocycler with the lid temperature set at ≥100°C, and perform PCR with the following cycling condition:

Step

Temperature

Time

Cylcles

Initial Denaturation

98°C

30 sec

1

Denaturation

98 °C

10 sec

5

Annealing & Extension

72 °C

30 sec/kb

 

Denaturation

98 °C

10 sec

5

Annealing & Extension

70 °C

30 sec/kb

Denature

98 °C

10 sec

25-35 *

Annealing

62 -70 °C **

15 sec

Extension

72 °C

30 sec/kb

Final Extension

72 °C

5 min

1

Hold

4 °C

 

*To determine the optimal PCR cycles, it can be useful to set up duplicate reactions, one with 20 cycles and one with 30 cycles.

**Annealing temperatures are determined by the primer sequences. Optimal results are observed with primers that have high Tms, preferably 68°C or higher. For Tm calculations, please visit: http://tmcalculator.neb.com.

2.3 Store the PCR product at -20°C or proceed directly to downstream applications. For example, to clone the PCR product, proceed directly to the NEB® PCR Cloning Kit (NEB #E1202). The PCR product can also be directly sequenced by Sanger sequencing after a PCR product cleanup step has been performed using Exo-CIP Rapid PCR Cleanup Kit (NEB #E1050).

 

General Guidelines:

1. RNA template

5´ RACE reactions require intact RNA template (total RNA or mRNA) in the range of 10 ng - 1 μg, preferably 100 ng -1 μg for total RNA or 10 ng -1 μg for mRNA. Lower RNA inputs are prone to non-specific amplification. It is not necessary to isolate mRNA from total RNA.

2. RT primer

For RNA targets with a poly(A) tail, optimal results are observed by using long anchored d(T) oligos, (e.g., d(T)40VN) as an RT primer. However, if desired, random primers or a gene-specific primer can be used in place of the d(T)40VN primer.

For RNA targets without a poly(A) tail (e.g., bacterial RNA), random primers or a gene-specific primer should be used for cDNA synthesis.

If a gene-specific RT primer is used during the RT step, it is best to use a gene-specific primer at least 10 nt interior to the RT primer during PCR.

3. Template switching oligo (TSO)

Many TSO sequences can be used successfully in this protocol. For optimal results, we recommend using a TSO that terminates with rGrGrG at the 3´ end. The following TSO sequence 5´ GCTAATCATTGCAAGCAGTGGTATCAACGCAGAGTACATrGrGrG, has been used extensively used during protocol development. TSO specific PCR primer sequence is underlined.

4. PCR primers

If the recommended TSO above is used, the underlined sequence, CAT TGC AAG CAG TGG TAT CAA C, can be used as the TSO-specific primer during PCR.

When designing the gene-specific PCR primer, a primer with a Tm greater than 68°C is preferred. For Tm calculations, please visit: http://tmcalculator.neb.com. If a gene-specific RT primer is used during RT, the gene specific PCR primer should be at least 10 nt interior to the RT primer.

5. PCR

Because of its superior fidelity and robustness, we recommend Q5 Hot Start High-Fidelity Master Mix (NEB # M0494) for the PCR amplification step.

Touch-down PCR is highly recommended. It tends to provide higher specificity and no downside effects have been observed.

Due to variable expression levels and template switching efficiencies, we recommend performing two PCR reactions (20 cycles and 30 cycles) to determine the optimal cycling number. For low abundant genes, up to 35 PCR cycles can be used.

6. PCR products

The PCR products generated using Q5 High-Fidelity 2X Master Mix have blunt ends and can be cloned using NEB PCR Cloning Kit (NEB #E1202S). If T/A-cloning is preferred, the PCR products should be purified prior to A-addition as Q5 polymerase will degrade any overhangs generated.

For some transcripts, multiple distinct PCR products may be observed due to alternative transcription start sites.

If non-specific PCR products are observed, please consider the following critical parameters for improvement. First, the RNA should be intact and free of contamination. Second, enough RNA template should be used. 100 ng-1 μg total RNA or 10ng – 1 μg of mRNA is preferred. Third, design of PCR primers is critical to achieve high specificity. Gene-specific primer with higher Tm, 68°C or higher is recommended. A nested PCR may also improve the PCR purity.