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oligo d(T) or random hexamer??

oligo d(T) or random hexamer?? - Real-Time PCR and Quantitative PCR Forum

oligo d(T) or random hexamer?? - Real-Time PCR and Quantitative PCR Forum


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Old 03-16-2009, 04:58 PM
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Default oligo d(T) or random hexamer??



Dear all,
I did qRT PCR with cDNA as the templates. I did two times, just for my curious, first for producting cDNA I used oligo d(T) and the other with random hexamer.
The problem is the qRT PCR data of both experiments are very different.
For example, by using cDNA with oligo d(T), the expression of the gene is shown high expressed (up regulated). But by cDNA with random hexamer, the gene is down regulated. I did just like normal qRT PCR (three replicates and three biological replications). So i'm really confuse now.
Is there anyohe who can explain it or perhaps has the same experiences??
When people should choice hexamer or oligo d(T) as the primer for cDNA production?

thanks for your answer
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Old 03-17-2009, 04:42 AM
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Default Re: oligo d(T) or random hexamer??

Hi
we prepare cDNA for qPCR using random hexamer as primers. we didnt compare the results using these two types of primers. they are working good.
a person who has already comare the two different types of priemrs for cDNA preparation can better explain this....
hope to get some good explaination for this.

regards
aftab
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Old 03-18-2009, 12:52 AM
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Post Re: oligo d(T) or random hexamer??

Quote:
Originally Posted by effendi View Post
Dear all,
I did qRT PCR with cDNA as the templates. I did two times, just for my curious, first for producting cDNA I used oligo d(T) and the other with random hexamer.
The problem is the qRT PCR data of both experiments are very different.
For example, by using cDNA with oligo d(T), the expression of the gene is shown high expressed (up regulated). But by cDNA with random hexamer, the gene is down regulated. I did just like normal qRT PCR (three replicates and three biological replications). So i'm really confuse now.
Is there anyohe who can explain it or perhaps has the same experiences??
When people should choice hexamer or oligo d(T) as the primer for cDNA production?

thanks for your answer
Good news is that you were smart to test both sets of primers.

Bad news is that they don't correlate; one up the other down-regulated.

Next check your PCR products on gel look for extraneous bands. Shouldn't be a problem--but nice to rule it out.

Then, did you use same concentration for oligo-T and hexamer during RT step.

Third use your PCR primer for the RT step. You can use the primer pair or just the Reverse primer---use it at low concentration, of course.
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