I would like to check my genes' expression by RT-PCR. cDAN was synthesized from total RNA.
Hi,
First of all, if you want to quantify gene expression the best way is qPRC. But if you do not have this option, before RT, you can normalize the samples including the same amount of RNA (ng) in each reaction, for example: 200 nf of RNA / reaction. Then, according to the RNA concentration, the volume in each reaction would be different.
Afther this, all your samples would have the same amount of cDNA, considering that RT reaction would be the same in each sample. Finally, you can make a PCR with the cDNA and electrophorese. I only recommend this if you want to test presence or absece of a product or a high reduction in your gene expresion. In other situation, qPCR is more accuarte.
In case you can make a qPCR, you can normalize your cDNA before RT and use the same volume / well in the plate. In the plate, it is important that you put the same volume of cDNA in the wells with target genes and referece genes in order to compare
Good luck
No. If you want to (semi)quantify differencial gene expression, you must use the same volumen of cDNA template and never adjust its concentration.
No, you don't need to do that! you adjusted the quantity of the material (sample) from you isolated RNA
You can use the same amount for each gene you study. The problem occurs when the concentration of cDNA is so high, that it can inhibit the reaction. But usually, it's difficult to have so high concentrations. I use from 10ng to 500ng, depending on the sample. You can check it. Try a qPCR with a reference gene and see the Ct. If it is to low 25, increase.
Actually i am asking this question in order to figure out how to normalize my samples. I am going to use my reference gene but how about if i get different band intense!
You need not adjust the concentration after synthesis of cDNA. But you need to adjust the con. of total RNA. before cDNA synthesis.
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