I am very new to quantitative PCR and need some help for this technique. For my experiment, I need to perform a quantitative reverse transcription PCR. I extract the RNA from my pellet and quantify it using NanoDrop. Afterwards, I need to convert that RNA into cDNA. I will use 1ug of RNA which I assume at the end of the thermal cycle, I will have 1ug of cDNA.
I have 6 samples and my house-keeping gene is GAPDH. I do not know what to do after cDNA synthesis. I will be quantifying them with Sybr Green.
My question is:
I need to do serial dilutions prior going to the last step of the quantitative reverse transcription PCR? if so, when do you make the serial dilutions? Once you have your cDNA?
If I have 1ug of cDNA, and I make serial dilutions. Do I have to make serial dilutions for all 6 of my samples? Example: If have to make 5 serial dilutions of, let's say... the following below:
-0.005ng/ul
-0.05ng/ul
-0.5ng/ul
-5ng/ul
-50ng/ul
Does that mean that it's 5 serial dilution of my control, 5 serial dilutions of my treatment, 5 serial dilutions of treatment/treatment, etc from the example mentioned above? Until I reach my 6th sample?
Do the serial dilutions also needs Sybr Green dye? or is it only your target genes?
I am very lost here with quantitative reverse transcription PCR.
Follow these discussions. You will get the best answers.
https://www.researchgate.net/post/is_it_necessary_to_dilute_cDNA_before_qPCR_How_much_dilution_should_be_made_How_to_determine_best_dilution_factor
https://www.researchgate.net/post/Why_is_necessary_to_dilute_cDNA_before_performing_PCR
https://www.researchgate.net/post/Is-cDNA-from-1ug-RNA-diluted-18-equivalent-to-cDNA-from-025ug-RNA-diluted-12
cheers!!!
A couple of points here.
1. Fix the amount of RNA that will be converted to cDNA (you have done).
2. Fix the amount of template that goes into the reaction vial by making a standard curve that you have mentioned. This is a 10fold dilution not 5 fold since the difference between consecutive terms in 10times. This also held to estimate the efficiency of qPCR Rx. Also, to fix the amount of template, as mentioned already. Depends on the sample whether to do with all or one. Yes, they do need SYBR.
3. Use a primer matrix to settle the optimum primer conc.
4. Use a nested qPCR design including a) Biological b) Sample and c) technical replicates. (MIQE guidelines)
5. Choose appropriate reference genes and the calibrator sample.
All the best!
Hi,
Just would like to add that you cannot assume that you will get 1ug cDNA from 1ug RNA since the efficiency of reverse transcription can vary.
https://reader.elsevier.com/reader/sd/pii/S2214753517302188?token=2B60E6E6DA880C6B2E736320360472FEE84377E4A618B08E0E0D8CADA7EE7A8AD37D7F7BF1E6CF7AC3FFB62A053FB90C
Hello, Eduardo dear
I have already read in an article that mentioned at least 10 ng/ul cDNA from each sample.
The important thing is that it should be the same concentration for all reactions, So that no error occurs during normalization.
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