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Workflow of qRT-PCR for siRNA


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5 replies to this topic

#1 dreww

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Posted 14 March 2010 - 06:26 PM

I have another post on siRNA against Beta actin. and i am gonna look at in vitro cDNA synthesis after a trizol harvest. i've read about qRT-PCR on siRNA, but need help on the workflow. Do i need primers specific to my siRNA strand(S+AS) or how does this work?

Any suggestions or help is greatly appreciated.

#2 pcrman

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Posted 14 March 2010 - 07:19 PM

why do you want to detect siRNA by qPCR or do you mean detecting beta-actin after RNAi?

#3 Dr Teeth

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Posted 15 March 2010 - 10:07 AM

I have another post on siRNA against Beta actin. and i am gonna look at in vitro cDNA synthesis after a trizol harvest. i've read about qRT-PCR on siRNA, but need help on the workflow. Do i need primers specific to my siRNA strand(S+AS) or how does this work?

Any suggestions or help is greatly appreciated.


Assuming you wish to detect the level of knockdown for B-actin using qRT-PCR following siRNA transfection of an siB-actin oligo, you will most likely need to:
1) Seed your cells to be transfected
2) Transfect your siRNA of interest
3) Wait 24-72 hrs for knockdown of your target
4) Harvest your cells using trizol and isolate total RNA
5) Perform cDNA synthesis using random hexamers, etc.
6) Perform qRT-PCR using primers against your target (B-actin) that are specific for an RNA target (i.e. cannot amplify from a DNA template)
and also using primers specific for an unchanging house-keeping gene
7) Calculate the level of B-actin knockdown by normalizing against the expression of the house-keeping gene

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#4 dreww

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Posted 18 March 2010 - 04:32 PM

Thank you Dr. teeth. that was my work flow. but instead of transfecting my siRNA, i got a vector to express my siRNA called pSilencer from ambion.
is there any way to detect the siRNA via qRT-PCR to make sure it's being expressed properly?

#5 pcrman

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Posted 19 March 2010 - 10:02 AM

yes you can detect the expressed siRNA in a way similar to detecting mature miRNA. Usually it is not necessary to do that. Your target gene knockdown is a good indicator of siRNA expression

#6 dreww

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Posted 20 March 2010 - 01:43 AM

Thank you very much. it's good to hear that qRT-PCR is the best way to quantify knockdown. thanks everyone!




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