1. How do I know if my bands are real?
  2. 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.

  3. Why do I have more than one band?
  4. 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.

  5. I have more than one (5' or 3') end. Does the brightest band indicate the most abundant RNA?
  6. 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.

  7. Should I use poly(A) or total RNA?
  8. In general, total RNA is an adequate template, but for some rare targets, poly(A) RNA yields better results.

  9. Why would I get no band at all?
  10. 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.

Five Ways to Improve Your RLM-RACE Reactions

  1. Use high quality, intact RNA.
  2. 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.

  3. Use an RNA sample in which your gene is expressed at high levels.
  4. 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.

  5. Do not exceed the recommended amount of input material in each step.
  6. 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.

  7. Optimize the annealing temperatures of both PCR steps.
  8. 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.

  9. Include the recommended controls.
  10. 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.