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cDNA amount in qPCR

cDNA amount qPCR concentration

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2 replies to this topic

#1 Dupasch

Dupasch

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Posted 09 June 2013 - 01:59 AM

Hey everyone!


i´m going to do some qPCR. so I´ve tried to find out the amount of cDNA i can put in my qPCR. since my GOI is expect to be very low expressed i thought of RT the max amount of total RNA the manufactor recomanded for the Kit and put 1ul of this cDNA in my qPCR. If i´m correct this would mean 50ng of cDNA in my reaction.
As far as i know the amount doesn´t realy matter as long as i take the same amount in every reaktion in my experiment. I will normalize against 3 Housekeeping genes measued in the same charge of cDNA, and calculated the relative expression.
  • This expression should not change when i measure the same samples with 5ng or 50ng in my reaction?
  • is 50ng to much cDNA for qPCR?
  • how can i compare my results with other results from the same GOI (no concentration of cDNA is mentioned in the papers)?

Somehow i have the feeling i´m missing something important...Posted Image
Thanks for every suggestion!!

#2 Shefali Desai

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Posted 09 June 2013 - 11:06 PM

I hope you have found out that you are using proper housekeeping geans for your normalization of your assay.House keeping should have same exprasion value at all time points.

#3 Dupasch

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Posted 09 June 2013 - 11:10 PM

Yes Shefali,
i measured these Genes earlier and they gave constant Ct´s Posted Image Do you have an idea to the questions i wrote?

Best wishes!






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