Hi there

generally speaking neat cDNA is too much for a sensitive technique like qPCR and the use of neat cDNA ( along say with too much primers) increases background signal : especially primer dimers

As a veteran of qPCR and to put this in some sort of general framework typically in the past if I have limited amounts of starting material and reverse transcribe from 100 to 200ng of total RNA if I dilute my final RT reaction to between 50ul and 100ul I will end up using about a 1:16-1:32 dilution of my cDNA in my relative gene expression qPCR as determined by a 1:2 serial dilution for preceding standard curve analysis. This dilution figure for cDNA ( or something approximating to this order of magnitude) also applies for low copy number genes regardless of how much starting material I have. Alternatively, for high copy number genes of if o have sufficient stating material to reverse transcribe from 1ug of total RNA I have typically used something like a 1:100 dilution of my cDNA in gene expression analysis. To be more specific :

1. For cDNA derived from high copy number genes and/or abundant starting material such that RT reactions are performed from 1ug of total RNA perform a 1:10 serial dilution of your cDNA from neat to 1:10,000 dilution

2. Pick an amount of cDNA that yields a Ct value of between 15 to 25 cycles although 10 to 35 is also OK providing the triplicates at each dilution are within 0.5 cycles and thus the R2 value in that part of the standard curve is > 0.98. 

3. By implication never take a dilution of cDNA where triplicates begin to diverge and correlation breakdown.  In this scenario I would typically end up using about a 1:100 dilution of my cDNA

4. Nevertheless pick a dilution of cDNA that is at the lower limit of this Ct window where triplicates are still concordant rather than a dilution of cDNA that also yields concordant data and falls within this ambient CT window. This is because at higher Ct concentrations you can potentially increase background and in particular formation of primer dimers

5. For low copy number genes or where starting material is limited such that you are obliged to reverse transcribe from (say) as little as 100ng of total RNA set up a standard curve by performing a 1:2 serial dilution from neat to 1:64 to 1:128.  Then follow the above guidance.  In these scenarios I typically end up using a 1:16 to 1:32 dilution of my cDNA for gene expression analysis

For General qPCR guidance including the above find attached a link to a stratagene guide in my drop box folder; in my 10 years of performing qPCR it is about the best guide I have come across 

https://www.dropbox.com/s/xfoig78a15yotkh/Introduction_to_Quantitative_PCR_web.pdf?dl=0

It is necessary to dilute the cDNA sample, since for most genes the cDNA is too concentrated for qPCR. Moreover, to calibrate a qPCR reaction for a new gene use a set of about 4 dilutions (for example 4 tubes of 3-fold or 4-fold dilutions) in order to determine the linear range of your reaction ( a range of concentrations in which each cycle doubles the DNA amount). Good luck.

I suggest 1 cDNA with 5 DEPC water.

Hi there

generally speaking neat cDNA is too much for a sensitive technique like qPCR and the use of neat cDNA ( along say with too much primers) increases background signal : especially primer dimers

As a veteran of qPCR and to put this in some sort of general framework typically in the past if I have limited amounts of starting material and reverse transcribe from 100 to 200ng of total RNA if I dilute my final RT reaction to between 50ul and 100ul I will end up using about a 1:16-1:32 dilution of my cDNA in my relative gene expression qPCR as determined by a 1:2 serial dilution for preceding standard curve analysis. This dilution figure for cDNA ( or something approximating to this order of magnitude) also applies for low copy number genes regardless of how much starting material I have. Alternatively, for high copy number genes of if o have sufficient stating material to reverse transcribe from 1ug of total RNA I have typically used something like a 1:100 dilution of my cDNA in gene expression analysis. To be more specific :

1. For cDNA derived from high copy number genes and/or abundant starting material such that RT reactions are performed from 1ug of total RNA perform a 1:10 serial dilution of your cDNA from neat to 1:10,000 dilution

2. Pick an amount of cDNA that yields a Ct value of between 15 to 25 cycles although 10 to 35 is also OK providing the triplicates at each dilution are within 0.5 cycles and thus the R2 value in that part of the standard curve is > 0.98. 

3. By implication never take a dilution of cDNA where triplicates begin to diverge and correlation breakdown.  In this scenario I would typically end up using about a 1:100 dilution of my cDNA

4. Nevertheless pick a dilution of cDNA that is at the lower limit of this Ct window where triplicates are still concordant rather than a dilution of cDNA that also yields concordant data and falls within this ambient CT window. This is because at higher Ct concentrations you can potentially increase background and in particular formation of primer dimers

5. For low copy number genes or where starting material is limited such that you are obliged to reverse transcribe from (say) as little as 100ng of total RNA set up a standard curve by performing a 1:2 serial dilution from neat to 1:64 to 1:128.  Then follow the above guidance.  In these scenarios I typically end up using a 1:16 to 1:32 dilution of my cDNA for gene expression analysis

For General qPCR guidance including the above find attached a link to a stratagene guide in my drop box folder; in my 10 years of performing qPCR it is about the best guide I have come across 

https://www.dropbox.com/s/xfoig78a15yotkh/Introduction_to_Quantitative_PCR_web.pdf?dl=0

Dear Laurence Stuart Hall,

Thank you so much for providing literature. However, due to some internet restrictions, I am unable to download provided material. I shall be very grateful to you if you please send this file on my email...

[email protected]

Hi Muhammad 

i I will send you that document

let me 

Dear Laurence Stuart Hall

With reference to your above stated answer,

What if I have large number of genes to study? For example 100 genes in 2 samples (each having 1 control and 1 treated). Would it not be nearly impossible to determine the suitable amount of cDNA by performing serial dilution method and amplification qPCR!

Dear Muhammed. Yes you would be right. You would either have to think about performing a cDNA reaction for each of your 200 genes with attendant optimisation such as cDNA dilution and efficiency of primer pairs from 200 standard curves as well as 200 expression assays. This would take you  at least 1 year !!! Alternatively with that many genes the easiest way might be to screen your RNA with a micro array and then verify by qPCR just that subset of your genes that showed significant changes from your array based data. There are other multiplex Taq man or array card assays you could try but these require specialist equipment and I cannot comment further as I am not intimately aaquianted with such assays. There are of course external companies that could do this work for you 

I have a follow up question on this: if I dilute my cDNA 1:5, then after I calculate my copy number, I multiply by my dilution factor correct? for example, if my slope was -3.29 and y-int : 14.8, and the Ct was 11.4, then copy number would be 7.44 (10^(Ct-Yint)/slope), but I had diluted the cDNA 1:5, so would that then make my copy number 37.22?

Thanks ( I know it is a dumb question but it's late on Friday and my brain is in shutdown mode

Yes. That is correct 

Of course you need to dilute it. I often use 1 microliter of cDNA from 20 microliter of total RT-PCR reaction volume, where the RNA volume was around 5 microliter of 5 microgram per microliter.

if you would like to study 100 of genes in two samples (C &T) is very easy, First u can construct  cDNA  using 2microgram of RNA with Life tech cDNA Kit (high fidelity), and dilute 1microL in to 20 microL DEPC water, then u can take 1microL of diluted cDNA for 3x10=30 reaction mix of qRT and make it easy, u will be get Ct values is19-28, melt curve will be 15-27. u can get very good amplification, melt curve and Ct values with out multiple amplifications peaks...

Laurence Stuart Dawkins-Hall I have one ore question. When the cDNA synthesis is done, (let say 20ul of cDNA at the end) first I add 80ul ddH2O to the cDNA and then I make the dilutions for primer efficiency. Is that correct? Otherwise the amount of cDNA is not enough. As you also said primer efficiency for each primer pair no matter it is GOI or housekeeping. Thanks in advance!

Yes

However with very low copy number genes it is sometimes an advantage to use neat cDNA but generally the pre dilution is best

Laurence Stuart Dawkins-Hall Thanks a lot!

Laurence Stuart Dawkins-Hall . I have some follow up.

Before performing the final RT-qPCR for single target gene

1. Find out the correct annealing temp for my experiment (using gradient )

2. Use the found annealing temperature to find out what dilution of cDNA to be used

3. Use the found annealing temperature and dilution factor for cDNA to validate the reference gene/genes that are suitable for my experiment.

My question number 1 is .. Is this pipeline correct ??

Second

Theoretically if I have 100 samples/cDNA (50 control and 50 treated), should I evaluate, annealing temperature and dilution factor, for each samples individually with triplicates OR can I pool the cDNA from all 100 sample and the perform that in triplicate.

1. Your pipeline is correct

2. Yes I would pool

Laurence Stuart Dawkins-Hall Vishnutej Ellur . Do the annealing temperature vary for each sample?. It should be primer specific right!!!. another doubt is, in RTPCR we wont use different temperature for each gene right?. Normally we use fixed 60C for all primers.

I use to design the primer using primer-blast and make sure they are having annealing temp close to 60C ( maximum difference between primers 0.5- 1 degree difference), it works perfectly fine to me. i get single peak in melt curve and no amplification in NTC. Please correct me if i m wrong.

And Laurence Stuart Dawkins-Hall thanks for the detailed answer.

Laurence Stuart Dawkins-Hall If you start with 100 or 200 ng/uL of total RNA to make 20 uL cDNA, how would you then dilute the cDNA with water to determine the most appropriate dilution of cDNA to use for the qPCR.

For example, let us assume you made 20 uL of cDNA, how would you do the dilutions from this step. Thanks for your input. When you run the various dilutions of cDNA, which Ct values would be most appropriate to choose when choosing the best dilution?

Also, do you need to dilute the cDNA, or can you use it as it is in the qPCR reaction?

With such a minimal amount, I would just dilute to 50ul or perhaps not at all and put 1ul into your qPCR reaction (where in effect it gets diluted)

I usually prepare the cDNA from 1ug total RNA in a reaction volume of 20ul and use 1ul (10X) cDNA for qPCR.

In the final reaction volume of 20ul cDNA, I add 80ul dilution buffer/ ddwater (for storing longer period), so its my 5X stock. Now whenever i need for qPCR, i take out 5ul from my cDNA stock (which is already 5X) and add 5ul ddwater to make final dilution of 10X and of course 10ul volume.

So anyway, think in terms of first principles. Let's say you use 1 ug of RNA in a 20 ul RT reaction, you're going to have 50 ng / ul of RNA in that reaction. Perform the RT reaction and then dilute to 100 ul, now per ul you have cDNA fro 10 ng of RNA.

As a very rough rule of thumb you can think that there is about 10 pg of RNA in a cell, so 10 ng is equivalent to RNA from about 1000 cells.

So this means that in 1 ul you have something like 1,000 copies of any transcript that occurred as 1 copy per cell. In my hands this will give a Cq of something in the order of 25. On the other hand if you have 10 copies of a transcript per cell you're going to have 10,000 starting RNA transcripts per ul, and this gives a Cq of about 20/21. Something like GAPDH can have over 1,000 copies per cell, and this would give a Cq of something like 14/15.

When you Cq gets very high you have to be thinking in terms of transcripts that are only expressed in a fraction of cells, but then you might have a heterogeneous cell population (for example I often do qPCR on mononuclear cells). But also when your Cq is high all sorts of stocastic effects come into play, and you have to start thinking about how your transcripts are Poisson distributed. For example if, on average, you have three transcripts of interest per reaction, then you have a 0.224 probability that your reaction will contain 3 copies of your target gene, and high probabilities that it will contain fewer than three, or more than three.