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standards for qPCR and some other doubts

qPCR standards cDNA

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9 replies to this topic

#1 Mad Researcher

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Posted 18 May 2015 - 01:19 PM

Hello,

 

1) I want to know how can i prepare standards for my qPCR. I will extract RNA from the brain followed by a DNAse treatment and the cDNA synthesis. I don't want to (if there are any) go for commercial available standards so kindly suggest how to prepare standards for qPCR and also please let me know if it is necessary to prepare standards?

 

2) What i know is i can make dilution series from my cDNA i.e. 1:1, 1:5, 1:25 and 1:125 and how do i decide which one to chose from the above dilution for further qPCR analysis?

 

3) I will use SYBR green master mix from thermo scientific. The protocol there says that my final vol. should be 20 ul. Can i reduce the volume or will it affect the qPCR?

 

4) Lastly, how can i decide the no. of cycles and the temperature or should i go for the builtin module? I am using stepone plus qPCR system.

 

Thanks :)


Cheers,

Mad Researcher

#2 bob1

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Posted 18 May 2015 - 08:23 PM

1)It depends on what you plan to do with your PCR - do you want copy numbers per sample, or are you doing relative quantitation?

 

2)Choose one, use consistantly.

 

3)Volume is dependent on the system used, 20 ul is recommended for 96 well format. Smaller volumes may also work, but make pipetting (consistently and accurately) increasingly hard for adding samples as the volumes decrease.

 

4) Many people use 40 cycles. In theory samples with one copy of a gene should have generated enough copies to detect via Ct at 35 cycles or less, but you want to be beyond this point as reactions are usually not 100% efficient.

 

You might want to check out the MIQE guidelines for qPCR, which will give you a few things to think about: Bustin et al., 2009. Clinical Chemistry, 55:4, 611-622



#3 Mad Researcher

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Posted 18 May 2015 - 10:47 PM

1)It depends on what you plan to do with your PCR - do you want copy numbers per sample, or are you doing relative quantitation?

 

2)Choose one, use consistantly.

 

3)Volume is dependent on the system used, 20 ul is recommended for 96 well format. Smaller volumes may also work, but make pipetting (consistently and accurately) increasingly hard for adding samples as the volumes decrease.

 

4) Many people use 40 cycles. In theory samples with one copy of a gene should have generated enough copies to detect via Ct at 35 cycles or less, but you want to be beyond this point as reactions are usually not 100% efficient.

 

You might want to check out the MIQE guidelines for qPCR, which will give you a few things to think about: Bustin et al., 2009. Clinical Chemistry, 55:4, 611-622

Thanks bob1 :)

 

1) I am doing relative quantification.

 

2) So, i could use 1:10 and make it a consistent dilution for all my qPCR assays?

 

3 and 4) Thanks for the explaination :)


Cheers,

Mad Researcher

#4 bob1

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Posted 19 May 2015 - 01:05 AM

1) In that case you don't need standards - all readings are relative to the reference genes used.

2) yes... but 1:10 might not be the ideal dilution, test a range and see what works for you



#5 Mad Researcher

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Posted 19 May 2015 - 02:31 AM

1) In that case you don't need standards - all readings are relative to the reference genes used.

2) yes... but 1:10 might not be the ideal dilution, test a range and see what works for you

2) Yes. I was just giving an example. I can make a dilution series like 1:1, 1:5, 1:25 and 1:125 and then decide which one works for me?


Cheers,

Mad Researcher

#6 bob1

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Posted 19 May 2015 - 12:59 PM

yes.



#7 Mad Researcher

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Posted 20 May 2015 - 09:29 AM

yes.

Hello again,

 

I forgot to ask one thing. Once i have run the qPCR with the above mentioned dilution, how do i decide which one worked the best for me?


Cheers,

Mad Researcher

#8 bob1

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Posted 20 May 2015 - 12:54 PM

Look at the Ct and work out which gives you the highest sensitivity. Ideally the majority of samples should have a Ct between 10-25 cycles, below that point and the efficiency comes into play, above and there is too much sample to be sensitive.



#9 Mad Researcher

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Posted 21 May 2015 - 09:57 AM

Look at the Ct and work out which gives you the highest sensitivity. Ideally the majority of samples should have a Ct between 10-25 cycles, below that point and the efficiency comes into play, above and there is too much sample to be sensitive.

Thanks. Bob, could you give me an example of how to make a standard curve for a qPCR (just for my knowledge).


Cheers,

Mad Researcher

#10 bob1

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Posted 25 May 2015 - 12:54 PM

Basically if you have a solution with a known concentration of pure template (e.g. plasmid DNA) you can work out the number of copies per volume by determining the molecular mass of the template and using the n= mass/molecular mass calculation.  You can then use this to make dilutions that should have a certain number of copies per volume, which can then be used to make the standard curve.

 

I am sure there are other ways to do this too, but this is how I have done it in the past (10 years ago).







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