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Calculation help

cDNA qPCR conversion calculations

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6 replies to this topic

#1 Mad Researcher

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Posted 19 May 2015 - 05:31 AM

Hello,

 

I am a bit confused.

 

I did RNA extraction and the yield is 17.5 ng/ul (its low). My final volume is 30 ul

Next i did a DNAse treatment.

Now, i want to do a cDNA synthesis followed by a qPCR.

For cDNA synthesis i am using Thermo scientific cDNA synthesis kit and for qPCR i am using Maxima SYBR green kit.

 

For the cDNA synthesis the kit says use 1 pg - 5 microgram (ug) of template RNA. I want to know how can i convert 17.5 ng/ul to 1 pg or 5 ug.

 

Next, for the qPCR master mix, it says use < or = 500 ng of template DNA. So, how much cDNA should i use.

 

Please let me know how do i make these calculations.

 

Thanks


Cheers,

Mad Researcher

#2 phage434

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Posted 19 May 2015 - 05:48 AM

Well, you have a sample that is 17 ng per ul. If you had 1 ul, then you would have 17 ng. Done. The main issue here is to use sufficiently small volumes of your RNA sample to have little or no effect on the RT reaction. For a 20 ul reaction (e.g.) then 1 ul is usually inconsequential.

 

An RT reaction will likely not produce 500 ng of DNA. You could measure it if you bother to purify the reaction, but I would not.  I would simply add 1 ul of the RT reaction (containing your cDNA) directly to the qPCR mix, again assuming the reaction volume is relatively large.



#3 Mad Researcher

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Posted 19 May 2015 - 05:59 AM

Well, you have a sample that is 17 ng per ul. If you had 1 ul, then you would have 17 ng. Done. The main issue here is to use sufficiently small volumes of your RNA sample to have little or no effect on the RT reaction. For a 20 ul reaction (e.g.) then 1 ul is usually inconsequential.

 

An RT reaction will likely not produce 500 ng of DNA. You could measure it if you bother to purify the reaction, but I would not.  I would simply add 1 ul of the RT reaction (containing your cDNA) directly to the qPCR mix, again assuming the reaction volume is relatively large.

So, i don't need to convert anything as the protocol says you should have 1pg? And if i take 1 ul then i have 17000 pg of my RNA? Am i correct in saying this?


Cheers,

Mad Researcher

#4 phage434

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Posted 19 May 2015 - 06:23 AM

Yes, correct. This sort of calculation should be nearly automatic for you. If it is not, you need to figure out why. A key idea is to make it a practice to carry units (liters, grams, moles) along with all of the numbers in  your calculations. Convert molar into moles per liter. Learn how to convert moles to grams when you know molecular weights. Learn how to convert % solution to grams per liter.



#5 Mad Researcher

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Posted 19 May 2015 - 06:57 AM

Yes, correct. This sort of calculation should be nearly automatic for you. If it is not, you need to figure out why. A key idea is to make it a practice to carry units (liters, grams, moles) along with all of the numbers in  your calculations. Convert molar into moles per liter. Learn how to convert moles to grams when you know molecular weights. Learn how to convert % solution to grams per liter.

So, for the qPCR, i can take upto 2 ng. Will that be sufficient?


Cheers,

Mad Researcher

#6 phage434

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Posted 19 May 2015 - 07:43 AM

I don't know where your 2 ng number came from. But in general, the template amount for PCR is quite unimportant. Often smaller amounts work better since the presence of any potential inhibitors is reduced. PCR exponentially amplifies, so halving the amount of template simply increases the number of required cycles by about one.



#7 Mad Researcher

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Posted 19 May 2015 - 08:29 AM

I don't know where your 2 ng number came from. But in general, the template amount for PCR is quite unimportant. Often smaller amounts work better since the presence of any potential inhibitors is reduced. PCR exponentially amplifies, so halving the amount of template simply increases the number of required cycles by about one.

So, i can take 1 ul of the cDNA for my qPCR and it should work well?


Cheers,

Mad Researcher





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