Reverse Transcription and RACE Support - Troubleshooting

Please see the following causes and suggestions:

Low cDNA yield can result due to several different reasons. Please see a few listed below:

• Poor quality mRNA: visualize total RNA on a denaturing gel to verify that the 28S and 18S bands are sharp. OD 260:280 ratio should be 1.7.
• Template degraded by RNase contamination: maintain aseptic conditions.
• Inhibitors of SuperScript® II RT may be present: remove inhibitors by ethanol precipitation of the RNA preparation before the first-strand reaction. Include a 70% (v/v) ethanol wash of the RNA pellet. Test for the presence of inhibitors by mixing 1 μg control RNA and comparing yields of first-strand cDNA.
• RNA preparation may have co-precipitated with polysaccharides: precipitate RNA with lithium chloride to purify RNA.
• mRNA concentrations were overestimated: quantitate the mRNA concentrations by measuring the A260 if possible.
• If using 32P isotope, it may be too old: use isotope less than 2 weeks old.
• Not enough enzyme was used: use 200 U SuperScript® II RT/μg RNA.
• SuperScript® II RT activity was decreased by incorrect reaction temperature: perform the first-strand reaction at a temperature between 37°C and 50°C.
• DTT was not added to first-strand reaction.
• TCA precipitations were performed incorrectly: adequately dry GF/C filters before immersion into scintillant.
• SuperScript® II RT was improperly stored: store at -20°C. Do not store the enzyme at -70°C.
• The reaction volume was too large: the reaction should be done in volumes less than or equal to 50 μL.

Please see the following suggestions:

• Low concentration of first-strand synthesis: starting RNA quality is poor or too low, reaction volume is too large, and/or primer concentration is too low or primers were not annealed to RNA properly.
• Low concentration of second-strand synthesis: RNAse H was not added or improper dilution to the second-strand volume was used.
• Low number of clones: there is most likely a problem with the adapter ligation/phosphorylation; use phiX Hae III fragments as a control; ligate adapters to it and phosphorylate to determine whether this is the problem.

Good laboratory practices are important for long fragment, one-step RT-PCR. These include using high-quality templates (pure, fresh, and intact) and fresh primer solutions. Optimization steps to consider include use of longer extension times and increasing template amounts. Learn more about RT-PCR reaction optimization and setup by visiting our reverse transcription educational resources.

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.

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.

Several factors are important for the best results:

1. The key factor for success is the quality of the RNA. RNA degradation is the most likely reason for failure to obtain a correct RACE product. We strongly recommend that you analyze a sample of your RNA on an agarose gel before starting to confirm RNA integrity. The use of RNaseOUT™ RNase inhibitor ensures RNA stability during various enzymatic reactions. If you are concerned about RNA stability, you may check the stability of the RNA after each enzymatic reaction (CIP, TAP, and ligation reaction) using agarose gel electrophoresis. Resuspend the RNA in DEPC water after enzymatic treatment in an appropriate volume (see pages 7, 9, and 11 of GeneRacer® kit manual) and check 1 μL on an agarose gel. Compare with the same amount of untreated RNA to check for degradation.
2. 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 one end.
   • 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.

3. If a smear in GeneRacer® 5′ primer negative control is visible, then we recommend the following:
   • Use a different 5′ primer, called the GeneRacer® 5′ primer. It is homologous to the slightly different site at the RNA Oligo but it generally gives less background compared to the original 5′ primer. Here is the sequence of the GeneRacer® 5′ primer: 5′ GCACGAGGACACTGACATGGACTGA. This primer can be synthesized and used in the 5′ RACE PCR instead of the original 5′ primer. It should reduce the background in RACE PCR.
4. If you see smears when performing the negative control using 5′ primer, the other negative controls (no template, GSP with template) were fine, and all reactions had the same PCR conditions, then:
   • If there is background when using the 5′ primer and template than you should subtract those bands/smear from the actual RACE reaction discarding it so not to confuse with the real RACE bands. The smear in that negative control will always be there because every cDNA has the binding site for that primer. So there should not be any major concern about the smear. If the actual RACE PCR works then the RACE band would outweigh the smear background.

Without optimization, nested PCR may produce no band, a single band, several bands, or a complicated pattern of bands (a smear). Smearing or failure to amplify could alternatively be caused by poor-quality RNA, or absence of the target in the RNA used for RLM-RACE. Ensure that pure, high-quality RNA is being used as your starting material.

• Multiple bands can be due to multiple initiation sites for transcription of the target gene or primer homology; try a new set of primers. If you are using inner and outer gene specific 5′ RLM RACE primers (for nested PCR), try using each one of them in a PCR with a gene specific 5′ primer and an aliquot of the RLM-RACE RT reaction as template.
• Perform the minus-TAP control; the minus-TAP control RNA should not yield the same PCR products as the experimental RNA you are using. No bands should be produced since RNA has either been dephosphorylated with CIP or it has an intact cap structure that can’t undergo ligation to the 5′ RACE Adapter.
• Optimize the PCR annealing temperature; the annealing temperature of the outer PCR is less critical and should be 55–65°C while the inner, nested PCR may need to be higher to achieve the required selectivity in the amplification. If the PCR fails to give the expected results, repeat the experiment using a higher (try 2°C higher) annealing temperature.
• If you have a longer target, the amplification cycle can be extended 1 min for each kilobase of target over 1kb.
• If you have a high GC-rich region and/or other regions of stable secondary structure in the RNA transcripts, you can try increasing the temperature of the RT reaction so that secondary structure effects can be minimized. The M-MLV RT included can be used at up to 50°C. Increasing the temperature of the synthesis reaction may facilitate read-through by the RT enzyme.