RT-PCR - PCR conditions optimization (Jul/22/2009 )
Hi fishdoc,
I have used oligo dt primers for Ist strand synthesis but for IInd strand synthesis I used gene specific primers.
I will do some more exercise on this and get back to you again
ThanX a lot........
-novagen-
Hi
Iam unable to amplify 1.4kb band through RT-PCR instead iam getting 400bp band. Do we get non-specific bands in RT-PCR
thanX
-novagen-
Do you have a positive control? I'm assuming you need the PCR amplicon for cloning as there's no other reason you'll want such a long product from RT-PCR.
I think you need to optimise your PCR. Check your primers, try different annealing temperatures, and most importantly check your elongation cycle as it looks like it might not be long enough to amplify 1.4kb. If possible, use gDNA instead, although I guess if introns present your product might be way bigger than 1.4kb so that you'll need different conditions for cDNA and gDNA.
Hope this helps.
oh, and answering your question, yes you can get non-specific PCR amplification from cDNA, that's why I think you need to optimise 
-almost a doctor-
almost a doctor on Jul 28 2009, 06:18 AM said:
Do you have a positive control? I'm assuming you need the PCR amplicon for cloning as there's no other reason you'll want such a long product from RT-PCR.
I think you need to optimise your PCR. Check your primers, try different annealing temperatures, and most importantly check your elongation cycle as it looks like it might not be long enough to amplify 1.4kb. If possible, use gDNA instead, although I guess if introns present your product might be way bigger than 1.4kb so that you'll need different conditions for cDNA and gDNA.
Hope this helps.
oh, and answering your question, yes you can get non-specific PCR amplification from cDNA, that's why I think you need to optimise
I think you need to optimise your PCR. Check your primers, try different annealing temperatures, and most importantly check your elongation cycle as it looks like it might not be long enough to amplify 1.4kb. If possible, use gDNA instead, although I guess if introns present your product might be way bigger than 1.4kb so that you'll need different conditions for cDNA and gDNA.
Hope this helps.
oh, and answering your question, yes you can get non-specific PCR amplification from cDNA, that's why I think you need to optimise
is 1.4kbp the complete cDNA for your gene of interest?
what is your rna? total or mRNA? is it clean?
have you checked the quality of the RNA on a gel? if it looks degraded (rRNA bands not crisp) your target might be missing the 5' end and so the PCR step will fail.
what RT enzyme are you using? superscriptIII? a cheap RT enzyme will yield fewer successful complete cDNAs.
with quality RNA and a high quality RT enzyme you should be able to amplify 4kb and less pretty easily.
a product of 1.4 kb will likely contain many errors in it if you amplify without using a high fidelity RT reaction.
-eldon-
Hi
Actually my genomic clone is about 3kb and cDNA is 1.4kb.I could happily amplify the genomic clone using extension time for 90 sec. So ,i have used similar conditions for amplifying cDNA but i couldnt do. then I have tried with different annealing temp.(55,60,62).Iam getting that 400bp band only and not 1.4kb band.
What could be the problem?
suggestions appreciated
-novagen-
eldon on Jul 28 2009, 06:44 PM said:
almost a doctor on Jul 28 2009, 06:18 AM said:
Do you have a positive control? I'm assuming you need the PCR amplicon for cloning as there's no other reason you'll want such a long product from RT-PCR.
I think you need to optimise your PCR. Check your primers, try different annealing temperatures, and most importantly check your elongation cycle as it looks like it might not be long enough to amplify 1.4kb. If possible, use gDNA instead, although I guess if introns present your product might be way bigger than 1.4kb so that you'll need different conditions for cDNA and gDNA.
Hope this helps.
oh, and answering your question, yes you can get non-specific PCR amplification from cDNA, that's why I think you need to optimise
I think you need to optimise your PCR. Check your primers, try different annealing temperatures, and most importantly check your elongation cycle as it looks like it might not be long enough to amplify 1.4kb. If possible, use gDNA instead, although I guess if introns present your product might be way bigger than 1.4kb so that you'll need different conditions for cDNA and gDNA.
Hope this helps.
oh, and answering your question, yes you can get non-specific PCR amplification from cDNA, that's why I think you need to optimise
is 1.4kbp the complete cDNA for your gene of interest?
what is your rna? total or mRNA? is it clean?
have you checked the quality of the RNA on a gel? if it looks degraded (rRNA bands not crisp) your target might be missing the 5' end and so the PCR step will fail.
what RT enzyme are you using? superscriptIII? a cheap RT enzyme will yield fewer successful complete cDNAs.
with quality RNA and a high quality RT enzyme you should be able to amplify 4kb and less pretty easily.
a product of 1.4 kb will likely contain many errors in it if you amplify without using a high fidelity RT reaction.
Hi eldon,
Thanx for the reply
1.4kb is the complete cDNA sequence of my gene of intrest.
I use total RNA for rt-pcr reaction.
I have run the total RNA on gel ,observed two major bands i.e 28s and 18s rRNA very sharply and no other bands were seen on gel.
I used oligo-dt primer, M-Mulv reverse transcriptase enzyme for Ist strand synthesis then for second strand synthesis I used taq DNA polymerase for amplification.
Can you please describe poor RNA quality fails to synthesize 5' end , as Iam newand not much aware about RT-PCR reactions .
ThanX a lot
-novagen-
novagen on Jul 28 2009, 08:27 PM said:
eldon on Jul 28 2009, 06:44 PM said:
almost a doctor on Jul 28 2009, 06:18 AM said:
Do you have a positive control? I'm assuming you need the PCR amplicon for cloning as there's no other reason you'll want such a long product from RT-PCR.
I think you need to optimise your PCR. Check your primers, try different annealing temperatures, and most importantly check your elongation cycle as it looks like it might not be long enough to amplify 1.4kb. If possible, use gDNA instead, although I guess if introns present your product might be way bigger than 1.4kb so that you'll need different conditions for cDNA and gDNA.
Hope this helps.
oh, and answering your question, yes you can get non-specific PCR amplification from cDNA, that's why I think you need to optimise
I think you need to optimise your PCR. Check your primers, try different annealing temperatures, and most importantly check your elongation cycle as it looks like it might not be long enough to amplify 1.4kb. If possible, use gDNA instead, although I guess if introns present your product might be way bigger than 1.4kb so that you'll need different conditions for cDNA and gDNA.
Hope this helps.
oh, and answering your question, yes you can get non-specific PCR amplification from cDNA, that's why I think you need to optimise
is 1.4kbp the complete cDNA for your gene of interest?
what is your rna? total or mRNA? is it clean?
have you checked the quality of the RNA on a gel? if it looks degraded (rRNA bands not crisp) your target might be missing the 5' end and so the PCR step will fail.
what RT enzyme are you using? superscriptIII? a cheap RT enzyme will yield fewer successful complete cDNAs.
with quality RNA and a high quality RT enzyme you should be able to amplify 4kb and less pretty easily.
a product of 1.4 kb will likely contain many errors in it if you amplify without using a high fidelity RT reaction.
Hi eldon,
Thanx for the reply
1.4kb is the complete cDNA sequence of my gene of intrest.
I use total RNA for rt-pcr reaction.
I have run the total RNA on gel ,observed two major bands i.e 28s and 18s rRNA very sharply and no other bands were seen on gel.
I used oligo-dt primer, M-Mulv reverse transcriptase enzyme for Ist strand synthesis then for second strand synthesis I used taq DNA polymerase for amplification.
Can you please describe poor RNA quality fails to synthesize 5' end , as Iam newand not much aware about RT-PCR reactions .
ThanX a lot
sorry that i have not used the M-Mulv reverse transcriptase, but i took a look at it online and think it may not be as good as Invitrogen's superscript iii.
how long and what temp. are you using for your 1st strand synthesis? this is a critical point in amplifying rare cDNAs. detection of rare cDNAs and anything longer than 1kb is sensitive to the enzyme and chemical conditions...if your rna is not completely denatured, the RT enzyme will sometimes not synthesize your gene of interest to completion. this becomes evident when probing cDNA libraries...if you are unfamiliar with such libraries, they are notorious for the large number of incomplete cDNAs that you must then try and complete.
superscript iii is excellent because it works equally well at 45C and a higher temp such as 50C (if you have too many non-specific bands) and the RT rxn can be done for 1 hour without appreciable loss of RT activity. how long is your RT reaction? 30 min at 45C?
you can have high quality rna, that contains intact mRNAs for your gene, but if the RT pol cannot extend from your oligo dT to the initiator met codon you will not amplify the gene...hence, once again i mention superscript ( and no i do not work for invitrogen, but i use their enzyme exclusively because of the consistency in amplifying rare or long cDNAs).
next question -> have you performed a northern to see what size your transcript actually migrates at OR if there are alternatively spliced forms? even though the 1.4kbp is theoretically the size, it may have very long poly A tails depending on the cell type in which it is expressed, or large 5' upstream elements that also contribute to stability/cell expression/intracellular localization, or it is spliced to the smaller size (400bp) that you are detecting etc...these can contribute to a difficult RT reaction with a generic RT pol...not in all cases...but frequent enough to cause these kinds of hurdles.
how much template do you start with? 100ng - 2 ug? 100ng is fine if you are dealing with an abundant gene...2ug helps if the transcript(s) is rare...you mentioned inducible expression...is it artificial induction and have you assessed the conditions that give you maximal induction of your gene? sometimes tightly regulated genes have rna's with short half-lifes and so are destroyed immediately following translation.
is your gene expressed in a specific cell type and if yes, can you separate that cell type or cellular zone so that when you collect RNA you are enriching for your gene of interest? do you know if your gene has multiple copies on separate chromosomes?
sorry for the questions, but there are a lot of factors that can be at play when dealing with hard to clone cDNAs....I've experienced many of them. althought i do not routinely do quantitative RT, I quite frequently do what you are doing, in that I amplify complete cDNAs from genes that contain WAY TOO many introns.
one thing you can do is compare oligo DT 1st strand synth to gene specific 1st strand synth. then perform your PCR with primers that will only amplify the 5' end (e.g. product size of 300 bp). i have had instances where oligo dT 1st strand synth resulted in complete cDNA such that i could amplify the first 300bp, but with the same sample using a gene specific primer could not. always chalked it up to weird secondary structure affecting annealing of the gene specific primer. i have also dealt with the scenario that oligo dT 1st strand synth worked well in that the cDNA was complete (~9 kb), but the 2nd stage PCR amplification failed to amplify the entire cDNA. in this situation, and, which could be similar in your case, is to perform the 2nd stage in parallel with 2 separate primer pairs - one primer pair for the first half of the gene, and a primer pair for the second half. if you utilize a restriction site in the middle of the gene where your first primer pairs reverse oligo overlaps with the second primer pair oligo, you clone the 2 frag's separately, and then piece them back together for a complete cDNA. our group had to do this with a huntingtin gene from a sea sponge...it took 16 different RT reactions and a lot of cloning, but we got the gene and without error. for a 1.4kb frag., you should not have to go to such extreme lengths.
i would try superscript iii and a Pfu turbo taq first...doing a 1hr RT reaction using 2 ug of total RNA. these enzyme combos will be both robust with high fidelity to perform well for a 1hr RT and a 2nd stage PCR of 40 cycles. if this works, and you still get a 400 bp band along with a 1.4kb band, then you might have more than one transcript or an annoying non-specific competing product...this can be overcome by doing touchdown PCR during the 2nd stage synthesis...your first few cycles have a Tm 10C above the primer pair, the next few cycles it drops to about 5C above, the next few cycles the Tm uses is that of the primers, and the final 10-15 cycles are 5C below the Tm. this type of PCR can be amazing with respect to removing non-specific amplicons.
-eldon-