FirstChoice™ RLM-RACE Kit with Manual, 1 Kit - FAQs

The GeneRacer method is designed to ensure that only full-length messages are ligated to the GeneRacer RNA Oligo and PCR amplified after cDNA synthesis. It is highly recommended that you clone your RACE products and analyze at least 10-12 colonies to ensure that you isolate the longest message. Many genes do not have only one set of transcription start sites but rather multiple transcription start sites spanning sometimes just a few or other times a hundred or even more bases. Cloning of the RACE products and analyzing multiple colonies ensues that you detect the diversity of the heterogeneous transcription start sites of your gene. It is also possible that you might obtain PCR products that may not represent the full-length message for your gene. PCR products that do not represent full-length message may be obtained because:

-RNA degradation after the CIP reaction creates new truncated substrates with a 5' phosphate for ligation to the GeneRacer RNA Oligo. Be sure to take precautions to ensure that the RNA is not degraded.
-CIP dephosphorylation was incomplete. Increase the amount of CIP in the reaction or decrease the amount of RNA.
-PCR yielded a PCR artifact and not true ligation product. Optimize your PCR using the suggestions described above.

RACE PCR artifacts or nonspecific PCR bands can result from one or more of the following:

-Nonspecific binding of GSPs to other cDNAs resulting in the amplification of unrelated products as well as desired products.
-Nonspecific binding of GeneRacer primers to cDNA resulting in PCR products with GeneRacer primer sequence on both ends of the PCR product.
-RNA degradation.
-Contamination of PCR tubes or reagents.
Note: Artifacts usually result from less than optimal PCR conditions and can be identified in negative control PCR.

Please see the following causes and suggestions:
Contamination by genomic DNA or an unexpected splice variant - Pretreat RNA with DNase I, amplification grade (Cat. No 18068015).
Design primers that anneal to sequences in exons on both sides of an intron or at the exon/exon boundary of the mRNA to differentiate between amplified cDNA and potential contaminating genomic DNA.
To test if products were derived from DNA, perform a minus RT control.
Nonspecific annealing of primers - Vary the PCR annealing conditions.
Use a hot-start PCR polymerase.
Optimize magnesium concentration for each template and primer combination.
Primers formed dimers - Design primers without complementary sequences at the 3' ends.

Please see the following causes and suggestions:

Procedural error in first-strand cDNA synthesis - Use high-quality RNA as a control to verify the efficiency of the first-strand reaction.
RNase contamination - Add control RNA to sample to determine if RNase is present in the first-strand reaction. Use an RNase inhibitor in the first-strand reaction.
Polysaccharide co-precipitation of RNA - Precipitate RNA with lithium chloride to remove polysaccharides, as described in Sambrook et al.
Target mRNA contains strong transcriptional pauses - Use random hexamers instead of oligo(dT) in the first-strand reaction, increase the temperature, and use PCR primers closer to the 3' terminus of the target cDNA.
Too little first-strand product was used in PCR - Use up to 10% of first-strand reaction per 50 mL PCR.
Gene-specific primer was used for first-strand synthesis - Try another set of GSP or switch to oligo(dT). Make sure the GSP is the antisense of the sequence.
Inhibitors of RT present - Remove inhibitors by ethanol precipitation of mRNA preparation before the first-strand reaction. Include a 70% (v/v) ethanol wash of the mRNA pellet. Note: inhibitors of RT include SDS, EDTA, guanidinium salts, formamide, sodium pyrophosphate, and spermidine.
RNA has been damaged or degraded - Ensure that high-quality, intact RNA is being used.
Annealing temperature is too high - Decrease temperature as necessary and/or use touchdown PCR.

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.

Here are our suggestions to optimize your RACE reaction:

-Use high-quality, intact RNA
-Use an RNA sample in which your gene is expressed at high levels
-Do not exceed the recommended amount of input material in each step
-Optimize annealing temperatures for PCR
-Include recommended controls

Alternative splicing, an alternative polyadenylation site, and alternative start sites can yield legitimate multiple bands. Sequencing will help to resolve any uncertainty.

If the reaction gives a specific product and the control reaction does not, your band is probably real. The only way to be sure is to sequence the product.

If you are performing either 5' or 3' RACE, you will need one gene-specific primer and if you are performing both 5' and 3' RACE, you would need two gene-specific primers. The primers should follow the rules stated below:

-50-70% GC content to obtain a high annealing temp (>72 degrees C)
-23-28 nucleotides in length to increase specificity of binding
-Low-GC content at 3' ends to minimize extension by DNA polymerase at non-target sites (no more than two G or C residues in the last five bases)
-No self-complementary sequences within the primer or no sequence complementary to the primers supplied in the kit, especially at the 3' end
-Annealing temperature greater than 72 degrees C—using primers with a high annealing temperature will help to improve specificity of your PCR

To check the RNA for integrity, analyze 500 ng of your RNA by agarose/ethidium bromide gel electrophoresis. You may use a regular 1% agarose gel or a denaturing agarose gel. For total RNA you should see the 28S and 18S rRNA bands. mRNA will appear as a smear from 0.5 to 12 kb. The 28S band should be twice the intensity of the 18S band. If you do not load enough RNA, the 28S band may appear to be diffuse. If you are using a denaturing gel, the rRNA bands should appear very clear and sharp. The 28S band should run at 4.5 kb and the 18S band should run at 1.9 kb.

RACE stands for Rapid Amplification of cDNA Ends. It is a method used to discover the 5' and/or 3' end of full-length transcripts. If partial sequence is known for a transcript of interest, RACE can help to elucidate the full ORF, 5' UTR, and 3' UTR sequences.

We recommend the following:
- Use high quality, intact RNA.
The FirstChoice RLM-RACE protocol works best with intact RNA as template. Partially degraded RNA may work, but the amount of available template will be reduced.

- Use an RNA sample in which your gene is expressed at high levels.
If your signal is absent or low, and you know that it is expressed in your sample, try using poly(A) selected RNA. Poly(A)Purist MAG is ideal for this enrichment.

- Do not exceed the recommended amount of input material in each step.
Each step in the FirstChoice RLM-RACE protocol is carefully optimized for a specific amount of carryover of reactants from the previous step. Do not exceed these recommendations.

- Optimize the annealing temperatures of both PCR steps.
The single most important variable in RLM-RACE is the annealing temperatures of the nested PCRs. The FirstChoice RLM-RACE Kit includes enough primers for 100 reactions. Experiment with different annealing temperatures to identify the optimal thermal cycle.

- Include the recommended controls.
If there is a question about the performance of the FirstChoice RLM-RACE Kit or the suitability of your RNA as template, do the recommended control reactions. It is especially important to make and use the gene specific control oligonucleotides as suggested to evaluate your RLM-RACE templates.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

On rare occasions, even though the target is present in your sample, you may not get a product. The TAP, CIP, and RNA ligation reactions require access to the 5' end. The secondary structure of specific mRNAs may prevent these reactions or make them very inefficient.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

In general, total RNA is an adequate template, but for some rare targets, poly(A) RNA yields better results.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

No. PCR strongly selects for the product that has the highest amplification rate in the PCR. Quantitative analysis methods such as RPA, qRT-PCR, or northern blotting must be used to answer this question.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

Alternative splicing (5' or 3' RACE), alternative polyadenylation sites (3' RACE), and alternative start sites (5' RACE), can yield legitimate multiple bands. Again, sequencing will resolve any uncertainty.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

If the reaction gives a specific product and the no-TAP control reaction does not, your band is probably real. The only way to be sure is to sequence the product.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.