How can I get the 4.5kb Human typeI procollagen alpha1 chain cDNA Via RT-PCR fr - Seeking Help (Nov/04/2005 )
Hi Cinab,
First of all, do you know in which quantity your transcript should be present in your sample? If you are looking for a transcript which is made only in low amounts, it can be painful. So making a high quality RNA extraction from a fresh sample is very important. The extraction of mRNA instate of total RNA can be helpful too.
If you have a rare transcript in your sample it might be needed to prepare more than one RT reaction of the same sample to get a product. What Supplier not tell, all RT (MMLV and AMV) have a low efficiency, that becomes more serious on rare transcripts with a high background.
For the RT reaction Superscript II or better III are good choices. Even though you can use both at 50°C, at this temperature the MMLV (Superscript is based on it) has only a short half live time. You can try to lower the temperature a little (46-48°C) and at Betaine to your RT-Mix in a final conc. of 2M. This may helps to over'come some strong secondary structures and has a little stabilizing effect on the MMLV at higher temps. If you want to use an MMLV, take a RNAse H- one, because they work better at elevated temps than RNAse H+ one. With MMLV H+ you can hardly work at 45°C.
(If you have any RT or PCR Kits from Qiagen the containing Q-Solution is Betaine.)
Another choice is the use of an AMV based RT such from Sigma or Roche. These one you can use up to 60°C, which have a working optima at 54°C. If secondary structure is the real problem, an AMV H- is the better choice than MMLV H-.
As an internal control to see if your RT reaction is working use primer for GAPDH or beta-actin.
For your PCR increase the number of cycles up to 40-45. If you just starting with a handful of cDNA, i think it is the better way instate of doing a nested PCR. By the way you can do a nested PCR as well.
Taq alone is unable to get a 4.5kb product (on a highly optimized pcr or with a plasmid as template it's possible). I suggest to use a LA Polymerase Mix (Taq/proofreading Polym.) from Takara, stratagen ... for testing your PCR. These Mix are much easier to handle than pfu, pfx, vent. I've never get anything amplified with pfx.
Like varius was said before, use pfu turbo/pfu ultra or Phusion (NEB, very good one and much cheaper) if high fidelity is needed. But keep in mind working with proofreading polymerase can be tricky, specially on larger fragments and without any experiences. So optimization is needed!!!
I would use a fixed annealing temp (54-59°C). Decreasing the temp within the first 10 cycles is only helpful if you get unspecific products.
Maybe one of this helps.
Dear Vairus and Qraylax:
Many Thanks for YOUR help, and I very appreciate your advices. I will try your recommendation and the RT-PCR reagents from Takara!
First of all, do you know in which quantity your transcript should be present in your sample? If you are looking for a transcript which is made only in low amounts, it can be painful. So making a high quality RNA extraction from a fresh sample is very important. The extraction of mRNA instate of total RNA can be helpful too.
If you have a rare transcript in your sample it might be needed to prepare more than one RT reaction of the same sample to get a product. What Supplier not tell, all RT (MMLV and AMV) have a low efficiency, that becomes more serious on rare transcripts with a high background.
For the RT reaction Superscript II or better III are good choices. Even though you can use both at 50°C, at this temperature the MMLV (Superscript is based on it) has only a short half live time. You can try to lower the temperature a little (46-48°C) and at Betaine to your RT-Mix in a final conc. of 2M. This may helps to over'come some strong secondary structures and has a little stabilizing effect on the MMLV at higher temps. If you want to use an MMLV, take a RNAse H- one, because they work better at elevated temps than RNAse H+ one. With MMLV H+ you can hardly work at 45°C.
(If you have any RT or PCR Kits from Qiagen the containing Q-Solution is Betaine.)
Another choice is the use of an AMV based RT such from Sigma or Roche. These one you can use up to 60°C, which have a working optima at 54°C. If secondary structure is the real problem, an AMV H- is the better choice than MMLV H-.
As an internal control to see if your RT reaction is working use primer for GAPDH or beta-actin.
For your PCR increase the number of cycles up to 40-45. If you just starting with a handful of cDNA, i think it is the better way instate of doing a nested PCR. By the way you can do a nested PCR as well.
Taq alone is unable to get a 4.5kb product (on a highly optimized pcr or with a plasmid as template it's possible). I suggest to use a LA Polymerase Mix (Taq/proofreading Polym.) from Takara, stratagen ... for testing your PCR. These Mix are much easier to handle than pfu, pfx, vent. I've never get anything amplified with pfx.
Like varius was said before, use pfu turbo/pfu ultra or Phusion (NEB, very good one and much cheaper) if high fidelity is needed. But keep in mind working with proofreading polymerase can be tricky, specially on larger fragments and without any experiences. So optimization is needed!!!
I would use a fixed annealing temp (54-59°C). Decreasing the temp within the first 10 cycles is only helpful if you get unspecific products.
Maybe one of this helps.
Hi Graylox,
I wonder the betaine you mentioned above is Betaine hydrochloride? Its role in RT reaction is to disrupt the secondary structure of the template RNA just like the glycerol or DMSO? Do this reagent really work in reducing the secondary structure of the RNA? I have outlined that my model RNA has a 66% GC content in the whole sequence, actually, GC content in any segment from this RNA is never lower than 65%?
I have added glycerol with a final concentration 20% in the RT system, but the result is not as good as claimed, My RT product amplified by Pfu from invitrogen is a smear? I add 5% final concentration glycerol in the PCR system!
Thanks for your patient and kind reply!
Hi Cinba,
Your right, betaine destabilizes GC-rich DNA by binding to AT rich pairs in the major groove or by binding the minor groove and increasing the hydration of GC pairs. On RNA it has a similar effect. Do not use betaine hydrochloride due to its low pH when dissolved in water. This low acidity will kill PCR reactions. You can buy a prepared 5M solution or the monohydrate from Sigma. If you have a difficult template, there is a lot of testing to do. There is no guaranty that one or another additive will work on a template. You can get good results with i.e. 2M betaine on one template, but totally fail on another one, where only DMSO gives results. So it's just testing. But most often betaine gives on GC-rich templates better results then glycerol or DMSO. You should add it to your PCR reaction too (maybe not the RT reaction is your problem, but PCR is). Usually 1.3M final conc. works well. But for both RT and PCR you should titrate the conc. of betaine in a range from 0.75M to 2.5M in 0.25M increments to obtain best results. Additionally DMSO up to 5% in the PCR may help too. Thats a lot of work and stuff you need, but worthy to kick off your problems.
If you use betaine you should lower your melting temperature to 92-93°C and your annealing temp by 1-2°C, because betaine change the melting temperature.
Once again with Invitrogens Pfx alone I've never get any product (I usually use Pfu Turbo). Pfx worked for me in mix with Taq to amplify a 9kb fragment only. So you can try a mixture of 5U Taq and 1U of Pfx for a 50µl PCR reaction. The enzyme conc. is pretty high, just as starting point. If it should work I think you can lower it.
By the way, I've never seen someone using betaine in a RT reaction, when using an AMV (Takara, Sigma,Roche), because of the increased temperature above 50°C you use to overcome secondary structures. I've just used it with MMLV. But you can give it a try, if you will use an AMV. So then please tell me if and how it works.
good luck
Your right, betaine destabilizes GC-rich DNA by binding to AT rich pairs in the major groove or by binding the minor groove and increasing the hydration of GC pairs. On RNA it has a similar effect. Do not use betaine hydrochloride due to its low pH when dissolved in water. This low acidity will kill PCR reactions. You can buy a prepared 5M solution or the monohydrate from Sigma. If you have a difficult template, there is a lot of testing to do. There is no guaranty that one or another additive will work on a template. You can get good results with i.e. 2M betaine on one template, but totally fail on another one, where only DMSO gives results. So it's just testing. But most often betaine gives on GC-rich templates better results then glycerol or DMSO. You should add it to your PCR reaction too (maybe not the RT reaction is your problem, but PCR is). Usually 1.3M final conc. works well. But for both RT and PCR you should titrate the conc. of betaine in a range from 0.75M to 2.5M in 0.25M increments to obtain best results. Additionally DMSO up to 5% in the PCR may help too. Thats a lot of work and stuff you need, but worthy to kick off your problems.
If you use betaine you should lower your melting temperature to 92-93°C and your annealing temp by 1-2°C, because betaine change the melting temperature.
Once again with Invitrogens Pfx alone I've never get any product (I usually use Pfu Turbo). Pfx worked for me in mix with Taq to amplify a 9kb fragment only. So you can try a mixture of 5U Taq and 1U of Pfx for a 50µl PCR reaction. The enzyme conc. is pretty high, just as starting point. If it should work I think you can lower it.
By the way, I've never seen someone using betaine in a RT reaction, when using an AMV (Takara, Sigma,Roche), because of the increased temperature above 50°C you use to overcome secondary structures. I've just used it with MMLV. But you can give it a try, if you will use an AMV. So then please tell me if and how it works.
good luck
Hi Graylox，
Thanks very much for your nice and patient guide and I appreciate your suggestion!
I will continue to try!
best regards!