The First Choice RLM Race is a kit designed to identify the true 5’ and 3’ ends of your cDNA of interest. The process of 5’ RACE is a more challenging procedure than 3’ RACE, with many more steps. For that reason, there is no reason to buy a kit if you’re only doing 3’ RACE, but this kit can help if you are having problems getting clean 5’ RACE products following available protocols.
Ambion’s 5’ RLM RACE first requires that you treat total RNA or poly A RNA with CIP to remove the free 5’ phosphates from contaminating rRNA, tRNA, genomic DNA or mRNA fragments. The RNA is then TAP-treated to remove the mRNA cap from the full length mRNA. Next, an adapter sequence is ligated to the 5’ end of the mRNA. The adapter will not ligate to RNA dephosphorylated in the CIP step, thus reducing background amplification of fragmented mRNA. Adaptor ligation is followed by a reverse transcription step and finally, nested PCR can be performed using primers that ligate to the adaptor and your sequence of interest.
The tricky part comes in designing gene-specific primers for this final nested PCR step. You will need to design a nested set of primers that prime around 150 or 200 bp from where you think the 5’ end of your sequence might be. This is a problem if you do not have an idea about where you sequence starts and stops or where potential introns may lie. It can also be a problem if you amplify too small of a piece (it will be impossible to evaluate on a gel) or if your piece is too large (it may not amplify at all). In the past, I have designed primers sets spaced about 100 bp apart to increase the odds that I will get a good-sized product for analysis on the first attempt (unfortunately, raising the total cost). Additionally, if you are working on a gene that has areas of homology to other genes in your organism(s) you must be careful to avoid those areas when you design primers, otherwise, you will inevitably get multiple PCR products. You can, however, gel purify and sequence those bands in order to determine which represent your cDNA of interest.
In contrast, the process of 3’ RACE PCR is much easier. First, you will run an RT reaction using your RNA and the 3’ adapter as a primer. Then, you move immediately into the nested PCR step. Again, this will require designing a set of nested gene-specific primers that prime somewhere within your cDNA. It is best if it is within 1 kb of the true 3’ end, however, longer products can still be reliably amplified using an extended-range polymerase. This has worked without fail for me. Check the literature or the internet and you will find plenty of protocols that provide the adapter primer sequences without requiring the use of a kit.
It is only necessary to generate average quality RNA (10 ug of total or 250 ng polyA) in order to use this kit successfully. Due to the CIP treatment step, I have found that you will still get a true PCR product even if you have genomic DNA or degraded RNA contamination. At worst, I have ended up with two bands, instead of a single band, after performing nested PCR. When this happens, I have sequenced both bands; one is the right product and one turns out to be garbage.
Ambion provides you everything you’ll need for 5’ RACE except your RNA, the gene-specific primers, control primers (also specific for your cDNA), phenol, chloroform, thermostable polymerase and a thermocycler. The manual that comes with the kit is extremely simple for the first time user and provides clear guidance for primer design. The included control primers and suggestions for designing others, are excellent troubleshooting devices, however, our lab has not had to resort to troubleshooting. This kit has worked reliably in our lab for 5’RACE using many different cDNA targets.
Julie Nemecek
Graduate Student
Medical Microbiology and Immunology
University of Wisconsin-Madison