Do you need to put the same quantity of RNA to do cDNA, but what you must do it is standardize in what final volume your cDNA must has. For example if you have 2 samples, one sample with a concentration of 1200 ng/uL of RNA and other with 1000 ng/uL. In the both samples you will need to take 2 ug of RNA, what in volume means that the first sample you will need 1,66 uL of RNA from that sample to do a cDNA with a concentration of 2 ug of RNA, while for the second sample you will need 2 uL of RNA to do the cDNA. As final step you will need to dilute both samples in a final volume of 8 uL. In the first sample you will need to dilute that sample in 6,34 uL of water, while the other sample you will need to dilute it in 6 uL of water. Did you understood?

In your case, you will diluted each sample in 10 uL of water, so in that case for the first sample in my example you will need to dilute that sample in 8,34 uL of water, while for the second sample you will need to dilute in 8 uL of water.

I hope that it helps you,

Pedro

Dear, Ana. You need to take the same quantity of RNA because your result depends from the number of molecules. For example, GAPDH fluorescence starts to rise at 24 cycle, fluorescence of another gene at 21 cycle(1-st sample)and 24 cycle (2 sample). If you have taken the same quantity of RNA for cDNA you can say that your gene is up-regulated(1-st sample) and at the control level (2 sample).

Yes, you need to have exactly same amount of RNA to make cDNA.

Here is an example to clarify:

Let's say you have 3 RNAs their concentrations are

A: 250ng/ul B: 500ng/ul C: 1ug/ul

And if you want to use 1ug of RNA to make your cDNA.

You should get 4ul from RNA from A, 2ul from B, and 1ul from C then convert them to cDNA.

I hope it helps, and good luck with experiment...

Hi Ana,

As the others have stated, you definitely need to normalized the amount of RNA used in your cDNA reactions if you are performing gene expression studies via qRT-PCR.

For example, if your cDNA kit requires 1 ug of RNA for each 10uL reaction, you must first convert each of your nanodrop readings into uL/ug.

I recommend creating an excel file, especially if you have 100 samples. In the first column A input your nanodrop readings. In column B, use equation =(1/$A)*1000 and drag down until a value is generated for each of your 100 samples. The above equation will calculate how many uL you need to pipette of each RNA sample in order to ensure you are adding 1ug of RNA into every cDNA synthesis reaction. In column C you can calculate how much water to add to each reaction. You can do this simply by adding up the volumes of all reaction components and subtracting that sum from the total reaction volume. The difference represents how much water you need to add. Your kit should tell you how much enzyme and/or reaction mix to use for a 10uL rxn,

For example, say for a 10uL reaction volume your kit says to use 1 uL of enzyme and 4uL of reaction mix, then for the first sample below you would use 4uL RNA, 1 uL of enzyme, 4uL of reaction mix reaction, and 1 uL of water for the first reaction. If you use equation =10-($B+5) in column C and drag, you can easily calculate how much water you need for each of your 100 samples.

Column A (ng/ul) Column B (ul of RNA) Column C (h20 needed)

250 =(1/$B)*1000 = 4 =10-($B+5) = 1

564 =(1/564)*1000 = 1.77 =10-(1.77+5) = 3.22

789 =(1/789)*1000 = 1.27 =10-(1.27+5) = 3.73

234 =(1/234)*1000 = 4.27 =10-(4.27+5) = 0.73

Hopefully, I could help and good luck with your 100 samples!

Thank you all for your answers!

I've learned that you don't need to put the same amount of RNA because the only thing that interests is the the difference between your endogenous control and the gene you are studying. For example:

Sample A: 100ng/ul of RNA

Sample B: 200ng/ul of RNA

To make the cDNA for both of the samples I use 10ul of RNA, in other words:

Sample A: I use 1000ng to the cDNA

Sample B: I use 2000ng to the cDNA

When the cDNA is ready and I make the qPCR I should obtain the following Cts, for example:

Sample A: endogenous ct: 24.69

gene I want to study: 28.01

Sample B: endogenous ct: 20.43

Gene I want to study: 23.75

In other words, I've learned that the concentration of RNA does not matter to make cDNA, because the only thing that matters is the difference between the endogenous and the gene that you are studying, and this difference must be always equal in the same group of samples and independent of the concentration of RNA.

Is it right or wrong?

The endogenous ct cannot vary so much (from 20.43 to 24.69 in your case), this is because every cycle of differences between the samples you will double the quantity of mRNA, so if you do not have a homogeneous endogenous ct, it indicates that your endogenous it is not a good one, at least. An accepted endogenous variation is around less than 1 cycle between samples, more than that your result it is compromised.

Dear,Ana. If you will publish your results you need to use the same quantity of RNA from the all 100 samples. If you compare only GAPDH and gene to study, you will have less information. But when you compare all samples, your data will be much more informative.You can do that only if you previously standardised your samples.

Hi Ana,

Yup I agree with Anastasia. It is much better to use the same amount of RNA for all 100 samples. Your resulting qPCR result will be much more informative: compare the fold change of your GOI between the samples, etc. And I also agree with Pedro, your ct for GAPDH shouldnt vary to much. If it does, might mean some of your upstream steps could have been compromised (RNA extraction, etc)

Mariana suggested one way of making your life easier, to add the same amount of RNA. Another way I suggest is to dilute all your samples into the same concentration, so that you can simply add the same vol. of RNA for all your samples for the RT step.