I've used one of the ExactStart kits from Epicentre and it was quite straightforward to generate full-length cDNAs in less than a day. The difficult part is deciding if you want to get your data the old-fashioned way (cloning, picking colonies, Sanger sequencing) or if you want to do it using a next generation sequencing approach. In addition to varying poly(A) tail lengths and spice variants you will likely have imprecise/diverse transcriptional start sites.

hi, it all depends on the facilities you have. I have experience with Invitrogen 5' and 3' RACE kit which worked well for 3'RACE and i used Clonetech kit for 5' RACE. The time for obtaining complete cDNA depends on your gene of interest. I suggest that try to isolate good quality RNA for RACE and primer designing also matters in the RACE amplification strategy. Good Luck.

HI Mark

Thanks for your reply. I have always wanted to learn the old fashioned cloning method however for time purposes and logistics it may be better for me to go with next generation sequencing as I would have access to this while for the cloning method i would need someone to teach me. Is there specific things one needs to consider when doing the next generation sequencing approach?

Hi Pragya

thanks for your answer. when you say the time for obtaining complete cDNA depends on your gene of interest what do you mean by this. does it depend on expression level of the gene of interest? Also how many primers does would usually test for 3' and 5' race experiments and does one design them as for standard PCR?

Hi Peter,

I have some experience with the First Choice RLM -RACE kit (formerly Ambion, now Life technology). It is quite straightforward. Quality of the RNA sample is critical and fresh preps are probably the best before the first step, particularly for the 5' RACE. 3' race will generally required only one amplification to obtain enough product for your sequencing. The 5' RACE is more challenging as you need to discriminate between real 5' end and artificial 5' end resulting from RNA degradation/shearing. Nested PCR will be generally required except for highly transcribed messager.

Good luck.

Bernard

Hi Peter. Definitely consult tech support at Epicentre (an Illumina company) while setting up your experiment. The main issue for setting up an Illumina-based experiment is that you must have a great deal of diversity in your sample, so you can't study just one gene. You might end up doing a transcriptome-wide study and then needing some bioinformatics help, etc.