RNA concentration for cDNA synthesis

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morkurbaga
morkurbaga's picture
RNA concentration for cDNA synthesis

 Hello all,

I am new at QRTPCR. I tried a lot of reactions by equaling the amount of cDNA concentrations of my samples to 100 ng but i got variable results. My friends suggest equaling the RNA concentrations before cDNA synthesis. At this time I equal RNAs but i think i used small amount? I used 150 ng RNA for total 20 ul RT working volume.  It means i used 7.5 ng/ul RNA per RT reaction.  I think i should use app. 150 ng/ul right? i couldn't make a standart curve with my cDNAs because the concentration of cDNAs and its dilutions are too dilute? 

And i have another question. Is the DNA contamination in total RNA isolation so important? Cant i use in cDNA synthesis? 
Thank you for all...

Sushma Dagar
Sushma Dagar's picture
 Hi, 

 Hi, 
I am not sure wether I understood your problem correctly or not. So you are first synthesizing cDNA from RNA. Generally we use 500ng RNA for cDNA synthesis but with 100ng also we get nice results. So may be you can try 100ng RNA per sample for 20ul volume.  once you get your cDNA, quantify it.  We always use equal conc. of cDNA for QRT. I don't know how you are equaling the cDNA conc. You can try one way. once you know your cDNA conc. convert the whole cDNA sample into 100ng/ul or less may be. Then you can easily use it for QRT reaction. Like I generallly uses 500ng cDNA conc. per QRT reaction and I convert my sample into 100ng/ul. So I add 5ul to each well. by this way, we can avoid variability.

and yes your RNA purity is very important for QRTPCR becuase if some contamination is there in your sample then your quantification will not be correct. But there is one way to avoid this. You can try designing your primers at exon-exon junctions. This will prevent amplification of DNA contaminant. But still I would suggest to work on your RNA isolation method to improve your RNA quality.

hcheung
hcheung's picture
For Q-RTPCR, we generally

For Q-RTPCR, we generally dilute the cDNA to 5 ng/uL and then add 4 uL of that (more pipettable) to each well so that the total cDNA added is 20 ng. We have also done 25 ng per well, but don't usually add more cDNA than that. Q-RTPCR is extremely sensitive.

Not sure by what you mean when you say that you can't make a standard curve. If you are doing SYBER GREEN real-time, it may be a problem with the primers, which is what I have found.

As for DNA contamination, if your primers include an exon-junction primer, then DNA contamination is less of an issue.