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Problems with validating primers and low expression genes


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#1 Fran24

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Posted 03 May 2010 - 01:27 AM

Hi,

I am relatively new to qPCR and having a few problems validating primer sets. I will be using the delta delta Ct method of analysis but want to check the efficiency of my primers first so need to set up a standard curve with serial dilutions of my template. I wondered whether the serial dilutions should be made of the RNA prior to reverse transcription. then made into cDNA and then this used for the curve, or if the cDNA should be made, for example with 100ng RNA, and then serial dilutions should then be made from this? Or will either method be fine?

Secondly, the genes I am interested in are expressed at relatively low levels (getting Cts consistently higher than 30), would it be worth increasing the amount of template RNA I put in the cDNA reaction? Currently I use 100ng RNA in a 20ul reaction mix to make the cDNA.

Thanks.

#2 Trof

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Posted 03 May 2010 - 02:37 AM

For efficiency testing you don't need to make a dillutions of RNA, diluting cDNA is just fine. Also delta delta Ct doesn't count with efficiency, so you may then use rather the Pffafl method, which takes into account efficiency as well.

I think you can try increasing the template. For low-expressed genes there isn't much of a choice. Just be sure your PCR is optimised.

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#3 Fran24

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Posted 03 May 2010 - 08:07 AM

For efficiency testing you don't need to make a dillutions of RNA, diluting cDNA is just fine. Also delta delta Ct doesn't count with efficiency, so you may then use rather the Pffafl method, which takes into account efficiency as well.

I think you can try increasing the template. For low-expressed genes there isn't much of a choice. Just be sure your PCR is optimised.



Thanks for your reply, will use dilutions of my cDNA to check the primer efficiency.

Would you be able to clarify what you mean by ensuring my PCR is optimised, with regard to increasing the amount of template RNA? Just want to make sure I set everything up correctly!

#4 Trof

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Posted 04 May 2010 - 04:45 AM

Would you be able to clarify what you mean by ensuring my PCR is optimised, with regard to increasing the amount of template RNA? Just want to make sure I set everything up correctly!


That you tried various primer nad Mg+ concentrations and Tm temperatures, and are sure that's the best you can get for your gene. Optimisation can lower your Cts, but if the problem is really low abundace of your target gene, then you can't do anything more than increase template.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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