Experimental design for RT-PCR - (May/23/2015 )

Hi All

 

Good day

 

I am designing gene expression analysis of transgenic plants. I have 5 types of transgenic plants, with 2 types of control plants. I want to study the expression of 4 genes with two reference genes. I need your help to design the RT-PCR experiment. how to form the standard curve? do I need to run about 3000 samples, including serial dilution for every gene and for every plant, in triplicate?

 

What kind of quantification are you interested in? Absolute or relative?

Thanks dear Mad Researcher for the response.

 

 

Actually I want to compare the expression levels of 4 target genes from my transgenic plants with those in the control (wild type) plants. I shall be using SYBR fast kit and bio-rad IQ5 machine.

awan on Sun May 24 03:51:29 2015 said:

Thanks dear Mad Researcher for the response.

 

 

Actually I want to compare the expression levels of 4 target genes from my transgenic plants with those in the control (wild type) plants. I shall be using SYBR fast kit and bio-rad IQ5 machine.

Ok. you are quantifying relatively. You should use a calibrator in each of your plate and for data analysis.

Ya, I have to quantify the increased or decreased expression.

 

Ok, does Calibrator mean a reference gene?

or

its the gene of interest from the control/ wild type sample?

 

Kindly guide. 

awan on Mon May 25 03:59:43 2015 said:

Ya, I have to quantify the increased or decreased expression.

 

Ok, does Calibrator mean a reference gene?

or

its the gene of interest from the control/ wild type sample?

 

Kindly guide. 

You can call a Calibrator as a reference gene but REMEMBER its different form Housekeeping genes.

 

Qiagen has defined Calibrator samples as: This is a reference sample used in relative quantification (e.g., RNA purified from a cell line or tissue) to which all other samples are compared to determine the relative expression level of a gene. The calibrator sample can be any sample, but is usually a control (e.g., an untreated sample or a sample from time zero of the experiment.

 

Basically, it can be a control from your experiment. When using the double delta Ct method for data analysis you need the calibration value for analysis. This would be an important control for measuring inter- assay variability that may occur when multiple samples are run on different plates.

Dear Mad researcher

 

Its so nice of you, I got the point. 

One last thing to be asked;

When I run PCR for 1 gene, the plate has 7 cDNA samples (my Transgenics/ treatments/ control). should I prepare the serial dilution for every sample and control, and it should be in triplicate. Or I choose the best concentration from the calibrator/ control and run only that concentration in triplicate.

 

 Thanks for the help.

awan on Tue May 26 04:40:25 2015 said:

Dear Mad researcher

 

Its so nice of you, I got the point. 

One last thing to be asked;

When I run PCR for 1 gene, the plate has 7 cDNA samples (my Transgenics/ treatments/ control). should I prepare the serial dilution for every sample and control, and it should be in triplicate. Or I choose the best concentration from the calibrator/ control and run only that concentration in triplicate.

 

 Thanks for the help.

Sorry for my late reply.

 

You don't need to run the serial dilution for all your cDNA samples. Running it once is enough. Yes, the serial dilution should be/preferably in triplicates (Duplicates is also fine but you should make a triplicate). Run all your samples and NTC in triplicates. This way you can see if there is any change or not.

 

Thanks :)

I am very grateful for the help.