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RNA Extraction and q PCR questions , please please help?


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#1 sun shine 411

sun shine 411

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Posted 02 May 2021 - 07:27 AM

I do have 3 conditions, Basal, drug A 1uM 6 hours, drug A 1uM for 24 hours.

I would like to confirm the results of gene expression after basal and treatments with a specific drug. So first, I have to do RNA extraction, cDNA synthesis and qPCR.  In other words, I want to make it clear there is, or there is no stimulation of the tested

gene because I got fold change between 1.2 and 2, and I know this is not enough in order to Conclude the effect of treatment

on X gene. So I decided to do quadruplicate of each condition- that means I will end up with

12 samples, I am confused is it better to plate in 6 well plate or 6 cm dish In order to have enough RNA? And after extraction of DNA

I will end up with 12 samples.,

 

I want to test 4 genes. So in qPCR, I will do it in duplicate then I will end up with 12×2=24 Samples.

 

12 samples need RNA extraction and cDNA synthesis ( table) 

 

Then to run qPCR in (96 wells)

 

 

 

My questions are

1-    IS this protocol will solve my drought regards the stimulation of genes (fold change)?

2-    Is it better to plate in 6 well plate or 6 cm dish In order to have enough RNA?

3-    2- do the biological quadruplicate are correct in this way?

4-    Do I distribute the technical duplicate samples and gene (yellow) correctly in 96 well plate for qPCR?

 

Thanks in advance. 

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