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RT-PCR


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16 replies to this topic

#1 novagen

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Posted 22 July 2009 - 06:23 AM

Hi all,

Iam stuchup with one basic problem
I could succesfully amplify my gene of intrest using gene specific primers from genomic DNA but unable to do it with Ist strand cDNA template.
what parameters do I need to optimize for PCR amplification.
I have used same conditions for PCR ampliofication for both genomic DNA and cDNA amplification.

Appreciate ur suggestions :P
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#2 fishdoc

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Posted 22 July 2009 - 06:27 AM

Hi all,

Iam stuchup with one basic problem
I could succesfully amplify my gene of intrest using gene specific primers from genomic DNA but unable to do it with Ist strand cDNA template.
what parameters do I need to optimize for PCR amplification.
I have used same conditions for PCR ampliofication for both genomic DNA and cDNA amplification.

Appreciate ur suggestions :P



Can you use more RNA template in you cDNA reactions? I have a colleague that is trying to measure expression of some genes, but they're apparently expressed in low amounts, because he had a hard time getting Ct values lower than 30. He ended up adding a lot more RNA to his cDNA reactions and was able to get his Ct values into the 20s.

Is (could) the gene you're trying to measure (be) inducible?

Also, what primer are you using for your cDNA synthesis reaction, gene specific? Hexamers? Oligo dT?

Prokaryotic or eukaryotic?

Edited by fishdoc, 22 July 2009 - 06:29 AM.


#3 novagen

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Posted 22 July 2009 - 07:27 AM

Hi

The gene I am trying is an inducible gene from a plant.
only after induction, I have isolated RNA and used sufficient template for the rxn
what else do I do.
The gene iam trying to isolate has got many introns
Do I need to change annealing temp for PCR amplification

ThanX
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#4 fishdoc

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Posted 22 July 2009 - 08:25 AM

Hi

The gene I am trying is an inducible gene from a plant.
only after induction, I have isolated RNA and used sufficient template for the rxn
what else do I do.
The gene iam trying to isolate has got many introns
Do I need to change annealing temp for PCR amplification

ThanX



Are you running any positive controls with a constitutively expressed gene to ensure the first strand synthesis is working? Like B-actin or rRNA?

#5 novagen

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Posted 22 July 2009 - 08:31 AM

Hi fishdoc,

I always run actin first and only then go for specific gene

Thanx a lot for the reply
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#6 fishdoc

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Posted 22 July 2009 - 08:36 AM

Hi fishdoc,

I always run actin first and only then go for specific gene

Thanx a lot for the reply



So, are you saying that your actin controls are all positive, but you inducible gene is negative from the same sample?

If that's the case, my first instinct is that either the primers for the cDNA synthesis are not amplifying your transcript if you're using gene specific primers. With oligo dT or hexamers, that shouldn't be a problem. The second thing would be that expression is too low to detect, even though you say you've induced it. How much RNA are you adding to your cDNA synthesis reaction? Have you tried adding more to see if you get a better signal?

#7 novagen

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Posted 22 July 2009 - 06:50 PM

Hi fishdoc,

thanx for the reply
could be the expression level is low.
but once I got the cDNA amplicon lower than 250bp which I am supposed to get at 1.4kb.what could that be?

Thank you
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#8 fishdoc

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Posted 22 July 2009 - 07:49 PM

Hi fishdoc,

thanx for the reply
could be the expression level is low.
but once I got the cDNA amplicon lower than 250bp which I am supposed to get at 1.4kb.what could that be?

Thank you



I'm sorry, but I don't follow what you're saying there. I've always tried to keep my RT-PCR amplicons pretty small, preferably below 500 bp, but usually 100-250 bp. Not sure if your amplicon size is a problem or not, but if 1.4 kb is what you're expecting to get, that may be the reason for the low yield.

#9 novagen

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Posted 23 July 2009 - 06:45 AM

Hi fishdoc,

Today i tried to optimize the pcr conditions for RT-PCR (which I previously told about 250bp band)
I have decresed the annealing temp and increased the amount of template(Ist strand cDNA)
I could see 2bands i.e 250bp and 400bp along with primer dimers.but my doubt is do we get nonspecific amplification in RT-PCR

thanX
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#10 fishdoc

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Posted 23 July 2009 - 06:57 AM

Hi fishdoc,

Today i tried to optimize the pcr conditions for RT-PCR (which I previously told about 250bp band)
I have decresed the annealing temp and increased the amount of template(Ist strand cDNA)
I could see 2bands i.e 250bp and 400bp along with primer dimers.but my doubt is do we get nonspecific amplification in RT-PCR

thanX



That depends on what your primers were for cDNA synthesis. If you used oligo dT or hexamers, there's a good possibility of non-specific amplification. If you used a gene specific primer for cDNA synthesis, there's much less chance of non-specific amplification. If you're concerned about the product, you could purify it and sequence to confirm.

#11 novagen

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Posted 23 July 2009 - 06:31 PM

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........
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#12 novagen

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Posted 28 July 2009 - 03:54 AM

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
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#13 almost a doctor

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Posted 28 July 2009 - 05:18 AM

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 :P

#14 eldon

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Posted 28 July 2009 - 06:44 PM

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 :lol:


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.

#15 novagen

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Posted 28 July 2009 - 06:59 PM

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
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