how to produce a high-quality cDNA template for RT-PCR - produce a high-quality cDNA template (Jan/25/2005 )
hello everyone!I want to isolate a ~2.5Kb cDNA from wheat .sinece I already know its sequence, a RT-PCR strategy is employed.but always I failed and the problem mostly may be the poor reverse transcription effiency.I've used the M-MLV(promega) and also super scriptIII(invitrogene) later and even added DMSO ( final concentration of 2.5%,in order to disrupt the secondary structyre of mRNA) . the reverse transcription reaction temprature of 37,42 ,55,60 has been tested extensively but without effect.
anxious to get your help!thanks a lot!
how did you know that there is something with your cDNA quality but not your RNA quality or PCR efficiency. which kit did you used when you isolate your RNA from wheat? what's your PCR system? what is your DNA polymerase? did your primer work very well? i recommend you use nested PCR in your case.
I've checked my RNA before taking RT reaction so I am sure of its quality.I ascribe my failure to the bad reverse transcription also because I had performed another two RT-PCR:one may bring a product of ~400bp and the other of >1.5kb.in fact,the former reaction was successful but the latter disappointed .basen on these ,I made my conclusion of that.so here ,I will be appreciated of your suggestions on how to improve the reverse transcription.
the 400 bp band showed up but not the 1.5 kb band. right? is the 400bp and 1500 bp band amplified from the same cDNA or not? if it's not, you can't conclude that the failure of the 1500 bp resulted from the poor reaction of your RT. it's probable that your PCR didn't work on the 1500 bp product. either your 1500bp primer or your pcr system is not good enough. you know, it's easy to amplify 400 bp ( especially when it's some houskeeping gene such as beta-actin,etc because of its richment in total RNA) even if your RNA is not good enough on the condition that your RNA has not been completely degraded. but it's not applicable to your 1500bp product especially when its RNA is rare in your total RNA. i think the reliable method to evaulate your RNA quality is either to calculate its A260/280 or run a gel and see if there is any DNA contamination or RNA degradation (the brightness ration of 28s to 18s should be 2 or so). may be your PCR condition is not optimized for 1500 bp and you should find the optimal annealing temperature, Magenisum concentration, and high-quality DNA polymerase ( i reommend EXPAND HIGH FIDELITY PCR SYSTEM from ROCHE). I reommmend RNeasy mini kit from QIAGEN for high quality RNA extraction and i have used it before for many times and never fail. as for the RT, you just follow the manual. the activity of SUPER script III may be decreased when you did RT at 55 or 60 centigrade.if you still believe that your failure resulted from RT and want to do RT at higher temperature, try the RT product from TaKaRa which performes well at 60 centigrade or higher.
in my opinion,your failure resulted from: firstly, low quality RNA but not RT; secondly, low quality primers for 1500 or 2500 bp; thirdly, low effiency DNA polymerase. so you should try your best to get high quality RNA at first. use the highest quality 1.5 ml tubes, pipettes and the other regents through all the steps. try to get the recommended RNA isolated kit and try nested PCR.
dear littlecell,I've read your suggestions and feel them helpful.thanks for your kind !now ,a modified RT reaction is in process ,in which many of your advice are adopted.I hope it sucess!at last,really thank to you again!