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qPCR lacks reproduceability


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#1 littleaxt

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Posted 12 August 2009 - 05:10 AM

Hi everybody,

I have a big problem I can not explain and need some advice. If I don't get the clue I can burry my PhD.
In a few words what I am doing:
My samples are liver samples of wild caught mice (Apodemus). I validated some comercial available reference genes that were originally designed for lab mice. I ended up with five genes to be used as reference genes(Rps18, Sdha, Canx, Actg, Pgk1).

For each sample I analyse I did two indipendend RNA extractions with subsequent RT, so I have to indipendend samples (A and B) per animal.
I run each gene (five RGs, one GOI) in triplicates with one no template control. First I anaysed all the A samples, now I'm doing the B samples. The result is horrible as you can see on the attached example.
For example the difference between the mean Ct-values of the example between sample A and B of animal 39 was:
GOI 1.41
RPS18 0.48
Sdha -2.43
Canx 2.82
Actg 1.99
Pgk1 2.96

Both runs were excellent, no byproducts, clear distinct melting peaks, good efficiencies (see attachedment). I have no clue why it doesn't work. Can someone help me or has any idea for this?

Could it be that the RT reaction transcribes the different genes with different efficiencies and that this happens just by chance? Or could it be that the liver tissue is so inhomogenous that the fractions of different cell types may have caused this result?

I'm more or less lost here and needs some advice from more experienced persons.

So, thanx to everyone how might have a look upon this!

Jan

Attached File  Dok3.pdf   24.69KB   194 downloads

#2 eldon

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Posted 12 August 2009 - 07:12 AM

Hi everybody,

I have a big problem I can not explain and need some advice. If I don't get the clue I can burry my PhD.
In a few words what I am doing:
My samples are liver samples of wild caught mice (Apodemus). I validated some comercial available reference genes that were originally designed for lab mice. I ended up with five genes to be used as reference genes(Rps18, Sdha, Canx, Actg, Pgk1).

For each sample I analyse I did two indipendend RNA extractions with subsequent RT, so I have to indipendend samples (A and ;) per animal.
I run each gene (five RGs, one GOI) in triplicates with one no template control. First I anaysed all the A samples, now I'm doing the B samples. The result is horrible as you can see on the attached example.
For example the difference between the mean Ct-values of the example between sample A and B of animal 39 was:
GOI 1.41
RPS18 0.48
Sdha -2.43
Canx 2.82
Actg 1.99
Pgk1 2.96

Both runs were excellent, no byproducts, clear distinct melting peaks, good efficiencies (see attachedment). I have no clue why it doesn't work. Can someone help me or has any idea for this?

Could it be that the RT reaction transcribes the different genes with different efficiencies and that this happens just by chance? Or could it be that the liver tissue is so inhomogenous that the fractions of different cell types may have caused this result?

I'm more or less lost here and needs some advice from more experienced persons.

So, thanx to everyone how might have a look upon this!

Jan

Attached File  Dok3.pdf   24.69KB   194 downloads

did you perform standard curves for each primer pair? you want to get the Ct values for the primers to fall around cycle 23-25.
what is your reference gene? you compare your targets samples to the reference. the RT reaction, if using oligo dT, should amplify all mRNAs with similar efficiency with the amount of product being indicative of mRNA abundance.

Edited by eldon, 12 August 2009 - 07:13 AM.


#3 noelmathur

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Posted 12 August 2009 - 10:40 PM

Honestly, even though I myself have performed this technique, never trusted it. We have a group down the floor, they print their own arrays and to verify the regulation obtained in the array, they use qRT-PCR. The primers they use in this reaction as essentially same as the probes on the printed array, yet they have difficulties confirming. They confirm in private that this is not a right technique but thats what their boss likes. They even have had results where certain gene is downregulated on the array and upregulated in the RT-PCR. Now, how do you explain that?
P.S. - They are not noobs, they have been doing it for more than 6 years now and have optimized protocols to death, yet such results..

Is there any other way, you can think of to prove your goal?

If you decide to stick with it, my advice would be run again. Run 4 times and take 2 best and think of an alternative method to validate these results.

#4 littleaxt

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Posted 13 August 2009 - 12:39 AM

Honestly, even though I myself have performed this technique, never trusted it. We have a group down the floor, they print their own arrays and to verify the regulation obtained in the array, they use qRT-PCR. The primers they use in this reaction as essentially same as the probes on the printed array, yet they have difficulties confirming. They confirm in private that this is not a right technique but thats what their boss likes. They even have had results where certain gene is downregulated on the array and upregulated in the RT-PCR. Now, how do you explain that?
P.S. - They are not noobs, they have been doing it for more than 6 years now and have optimized protocols to death, yet such results..

Is there any other way, you can think of to prove your goal?

If you decide to stick with it, my advice would be run again. Run 4 times and take 2 best and think of an alternative method to validate these results.



Thanks Eldon, thank you Noelmathur!

I love your honest statement Noelmathur. Actually I think I know what went wrong: The sox, I have to wear the same sox every day, then it works. Sorry to my lab-mates who will suffer from this, but we all have to take our share.

No, maybe I have found the solution to it. No explanaition but a solution. So far I never had a problem with reproduceability when I re-ran a sample. Most of the time it was more or less in a short time frame so I used the same lot had same weather conditions, same moon phase whatever and the PCR produced consistent results.
When I now analysed the A and B samples there was a long time span in between. The weather changed, different sox etc. Yesterday I re-ran the samples I attached as an example in the same run and they matched quite well. Maybe this is the solution. Means a lot of work, as I have to repeat everything and I thought the PCR reaction itself would be more reliable but if this helps....

I would be very gateful if anyone has more suggestions :-)

Thank you for your help!

Jan

#5 noelmathur

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Posted 13 August 2009 - 12:57 AM

sox? do you mean socks? Sorry, I don't get it, you have got lucky pair of socks? :lol:

#6 littleaxt

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Posted 13 August 2009 - 01:29 AM

sox? do you mean socks? Sorry, I don't get it, you have got lucky pair of socks? :lol:


Sorry, yeah socks, of course...
Oh, you did not know? PCR depends strongly on the socks you wear. You should tell your colleagues.
At least this is my impression now dealing with real-time PCR....




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