The 5’RACE System, an acronym for “Rapid Amplification of cDNA Ends”, from Invitrogen is designed to amplify the unknown 5’ end of a messenger RNA template using a defined internal site. This is a well-defined system that is relatively easy to use. The system comes with all the necessary reagents and enzymes, except Taq polymerase for the final PCR amplification step. The user also has to design two internal primers within the known sequence for the reverse transcription and the final amplification.
The basic methodology of this system starts with prepared mRNA (in my experiments I was using bacterial mRNA). The quality and quantity of mRNA is a definite limiting step for this kit since mRNA is difficult to maintain and isolate. The protocol calls for 1-5ug of mRNA and 2.5pmoles of GSP1. GSP1 is the user defined primer that is approximately 300bp from the putative 5’ end of the mRNA to be used in the first strand synthesis of cDNA. The mRNA is reverse transcribed using SuperScript II RT enzyme for 30-50 minutes at 42C. The remaining RNA is removed using RNase for 30 minutes at 37C. The reaction is then purified using a S.N.A.P column with the system’s wash buffer and EtOH. The cDNA is eluted with distilled water. The cDNA is then tailed with a polyC tail using the tailing buffer, dCTP and TdT. Finally, this tailed cDNA is PCR amplified using a user defined nested primer (GSP2) and the system provided universal primer AAP that anneals to the polyC tail. The PCR product can then be visualized via agarose gel electrophoresis and sequenced using a nested primer. The system also provides an additional primer for nested amplification of the final product if the yield is low. There are defined stopping points in the protocol at which point the reaction can be stored at -20C. There are also control reagents, RNA and primers to check the protocol.
In my experience with the RACE system, I found it relatively easy to use however I have yet to get a concise sequence from my PCR product and usually visualized multiple bands via electrophoresis. The system manual is very detailed with pages of references and a very informative trouble shooting guide. Most likely, my troubles are due to the low concentration of starting mRNA for my gene of interest. However, the multitude of steps in the protocol leaves a lot of room for error. I have tried all of the troubleshooting techniques in the manual as well as consulted the technical support at Invitrogen. The support team was very helpful and maintained contact until they felt they had exhausted their expertise as well. Furthermore, the system does not give enough S.N.A.P purification columns and RNase in comparison to the other reagents and enzymes. I am not sure how this system can be improved since most of my problems with the experiment can most likely be attributed to my gene of interest. However, I have spoken to other colleagues that have used this system who also did not get adequate results. I have been able to ascertain a start site from the results but the sequencing is far from perfect.
Overall, the 5’RACE system does the best with what it is given. The process itself is tedious with lots of room for error. If a researcher is looking for the 5’ end of a gene of interest, this system is well worth the investment. However, it can be very time-consuming and difficult to achieve an adequate sequence.
Katrina Williams
Lab Specialist, Sr
Division of Infectious Disease
McGuire VA Medical Center
Richmond, VA