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Experimental design for RT-PCR

PCR gene expression molecular biology genomics

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6 replies to this topic

#1 awan

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Posted 23 May 2015 - 06:38 AM

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?

 



#2 Mad Researcher

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Posted 23 May 2015 - 01:00 PM

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


Cheers,

Mad Researcher

#3 awan

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Posted 23 May 2015 - 07:51 PM

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.



#4 Mad Researcher

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Posted 24 May 2015 - 08:59 AM

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.


Cheers,

Mad Researcher

#5 awan

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Posted 24 May 2015 - 07:59 PM

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. 



#6 Mad Researcher

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Posted Yesterday, 07:58 AM

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.


Cheers,

Mad Researcher

#7 awan

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Posted Yesterday, 08:40 PM

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.







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