RT-PCR for large fragment - (Aug/24/2005 )
Hi all
Is it difficult to amplify 3.5 kb fragment from cDNA?I am able to amplify this fragment in smaller overlapping fragments from the same cDNA using different internal primers specific for this gene but if using the very first forward primer and the last reverse primer I am not able to amplify the whole gene.I am using touchdown PCR.Is touchdown PCR not suitable for large fragment.Though I tried the specific taq polymerase also which can amplify upto 5kb.
Thanks
It is possible. In our lab, we regularly do RT-PCR amplicon is 3,8 kb, and we do this on HIV, and go to a copy number as low as about 500 RNA copies/ml plasma (which means in RT reaction we go up to about 100 RNA copies, and probably even less if you think that during purification you loose some RNA).
We do this with Roche's expand RT (just following the protocol) followed by nested PCR with Roche's expand high fidelity. I'm also very succesfull in doing this using Invitrogen's superscript III (of superscript III one step), followed by an inner PCR with platinum taq hifi of PfuUltra.
I do my inner PCR touchdown as well, but for the outer I haven't tried it.
If possible: optimise your PCR reaction (on plasmid of genomic DNA), and then proceed to the RT-PCR reaction.
We do this with Roche's expand RT (just following the protocol) followed by nested PCR with Roche's expand high fidelity. I'm also very succesfull in doing this using Invitrogen's superscript III (of superscript III one step), followed by an inner PCR with platinum taq hifi of PfuUltra.
I do my inner PCR touchdown as well, but for the outer I haven't tried it.
If possible: optimise your PCR reaction (on plasmid of genomic DNA), and then proceed to the RT-PCR reaction.
Hello,Vairus!
Now, I also RT-PCR the cDNA of Human procollagen typeI Alpha1 chains. The Full length of this cDNA fragments is 6.7kb, I just want to amplify the CDs of this cDNA(4.5kb). I have extracted the Total RNA from huamn skin, and electropheresis show The total RNA is intact! I, however, cannot get the fragments desired! the GC content of this fragment is 63%, and the Td predicted by Oligo is 110 centigrade degree! I PCR it With glycerol with final concentration 5%, but ,nothing is detected via Argrose Gel electrophoresis! Now I am very Upset! Can you give me some recommendations! Thanks!
What are your exact PCR conditions? What's the anenaling temperature you're using? Which RT, which conditions, what's the concentration of your RNA?
Any control PCR reactions you have that always worked?
Have you tried replacing your reagents, using new dNTP's, buffer, primer, enzyme...?
Any control PCR reactions you have that always worked?
Have you tried replacing your reagents, using new dNTP's, buffer, primer, enzyme...?
Hi Vairus,
My PCR conditions is melting at 94 centigrade degree for 3min, then 10 cycles, melting at 94 centigrade degree for 20Sec, anealing at 62 centigrade degree for 30sec, elongation at 68 centigrade for 5min, and 20cycles, melting at 94 centigrade degree for 20Sec, anealing at 65 centigrade degree reducing 0.5 centigrade degree each cycles for 30 sec, elongation at 68 centigrade for 5min, entension at 68 centigrade for 15min.
Oligo6 show that the Tm of my PCR product is110centigrade degree, should I improve my melting tempature from 95 centigrade degree to 110 centigrade degree?
RT condition is: reverse transcriptease is :MMLV which has RNaseH activity and at 37 has its full activity, MMLV is contained in the Kit provided by BBI. I proceed the RT reaction at 45 centigrade degree for 1.5Hour, because of the length(CDs:4.5kb, full lenth of the ProIα(1): 6.8Kb) and GC rich content(65%) of my fragment, I do it according to the guide of the protocol attached with the kit provided by the provider--BBI. I prime the mRNA with oligo(dT) and GSP(gene specific primer). I am sure my reagents has no problem!
should I continue to improve RT reaction Temprature? to what centigrade degree?
I do not adopt the control reaction, What mRNA affluent enough in Human skin could be used as a control ? how can I optimize my RT-PCR to get my product?
by the way, in the attached file is my RNA and cDNA secondary structure, please help me to analyze it . I await your reply! and I will be appreciate your suggestion! I am much owned to you for your help!
Hi there,
The Tm of your primers is very high, but when calculating Tm, how many base pairs do you include? It's only the first 6-10 that are really important to determine your Tm. Try lowering your Tm with for instance 10°C and if you still don't get any result (even
aspecific) then probably your RT reaction is the problem.
Have you tried performing the PCR on DNA? 6,7 kb is not so hard to PCR. Once optimised, you can check your RT reaction itself. I know this takes longer but it gives you better results in the end.
I don't see no reason to increase your annealing temp after 10 cycles. A higher annealing temperature will give you more specific results if you use it in the first steps and then decrease your annealing temp stepwise to increase your product yield. (the idea behind this is that at a higher temperature you will only get the specific product, and after a couple of cycles you will have that much specific product that even when you lower the annealing temperature the aspecific products will hardly get formed, but increasing temperature makes no sense).
I'm not familiar with MMLV RT, but as you have to use it at 37°C, are you sure it still has enough activity @ 45°C?
Considering the secondary structure of your RNA: I would search for an enzyme that can stand higher temperatures. Or do you first denature your RNA for 5 or 10 minutes @ 65°C? RT's that can stand higher temperatures are available from Takara, Roche, Invitrogen (and probably several others as well).
I am not doing RT-PCR on human tissue RNA, so I can't think of any positive control RNA, but search for it on pubmed or so...
Good luck!
The Tm of your primers is very high, but when calculating Tm, how many base pairs do you include? It's only the first 6-10 that are really important to determine your Tm. Try lowering your Tm with for instance 10°C and if you still don't get any result (even
aspecific) then probably your RT reaction is the problem.
Have you tried performing the PCR on DNA? 6,7 kb is not so hard to PCR. Once optimised, you can check your RT reaction itself. I know this takes longer but it gives you better results in the end.
I don't see no reason to increase your annealing temp after 10 cycles. A higher annealing temperature will give you more specific results if you use it in the first steps and then decrease your annealing temp stepwise to increase your product yield. (the idea behind this is that at a higher temperature you will only get the specific product, and after a couple of cycles you will have that much specific product that even when you lower the annealing temperature the aspecific products will hardly get formed, but increasing temperature makes no sense).
I'm not familiar with MMLV RT, but as you have to use it at 37°C, are you sure it still has enough activity @ 45°C?
Considering the secondary structure of your RNA: I would search for an enzyme that can stand higher temperatures. Or do you first denature your RNA for 5 or 10 minutes @ 65°C? RT's that can stand higher temperatures are available from Takara, Roche, Invitrogen (and probably several others as well).
I am not doing RT-PCR on human tissue RNA, so I can't think of any positive control RNA, but search for it on pubmed or so...
Good luck!
Hi Varius!
First of all , I appreciate your advises!
Because the Genomic DNA is too long, about 25kb, SO we decided to amplify its cDNA, You misunderstood what I mean at the anealing temprature, after 10cycles the anealing temprature has been decreased by 0.5℃ each cycles from 65℃, you mean I should lower my anealing Temprature？I would try!
MMLV's activity has been damaged by the high temprature, but according the provider's guidlines, I put the reaction system at 45℃, at the midway of the RT reaction, I add some MMLV enzyme! I completely obeyed the protocol offered by the BBI, I denature the model RNA at 75℃ after adding the primer but prior to adding the RNAse inhibitor and MMLV enzyme Mix. This RT-PCR really kills me! Thanks for your guide and help! I am a novice ! and I am a postgraduate at Graduate School of Chinese Academy of Sciences. My Email address: Dajiangc@yahoo.com.cn, I hope we could contact and discuss further! Many Thanks!
No other ones are willing to reply?
I am confused now，Please help me to solve this problem! Many thanks!