keeping total RNA input costant - (Nov/11/2005 )
I'm wanting to do an RT-PCR dilution series of my target gene, 1x, 2x, 5x 10x etc.
I also want to keep the total RNA constant to control things as much as possible.
Any ideas on different sources of RNA one can use to "bulk up"?
I've though about other animals and the obvious problems with sequence similarities esp 18S and so on.
Plant RNA?
Yeast RNA?
tRNA?
thanks in advance
I don't understand exactly what you are trying to do? Could you explain a bit more fully, please? why would you want to "bulk up" your RNA? wouldn't this invalidate any RT-PCR data you were able to obtain?
I want to do a linear dilution of my gene of interest to see the detection limits of the qPCR reaction.
As I dilute my target RNA, I want to dilute it in another RNA species that will not contain my GOI, therfore keeping the total RNA concentration constant between reactions.
why?
your detection limit will be based on copy# of GOI
why does it matter how much total RNA is present? the relative amount of that component in the reaction is pretty small and should not effect your detection limit. I would worry more about reaction efficiency at low copy numbers.
perhaps I am wrong, but I do not think that it matters to keep the total RNA the same.
good luck
you also have to look at another gene to ensure the input RNA is the same. Somthing that won't change with your treatment, like a housekeeping gene. using the gene that doesn't change with your treatment is how you ensure that that amount of input RNA is the same. you can then normalise your goi against the housekeeper, to get more believable results.
vetticus
If all you care about is the qPCR, why not dilute the cDNA after RT?? Do a no RT reaction to add instead of water if you want to have the same amount of everything... (I guess everything except DNA...)
Are you using primers that span an intron? maybe you could add gDNA???
Hope this helps...
I also want to keep the total RNA constant to control things as much as possible.
Any ideas on different sources of RNA one can use to "bulk up"?
I've though about other animals and the obvious problems with sequence similarities esp 18S and so on.
Plant RNA?
Yeast RNA?
tRNA?
thanks in advance
I did something similar. I got my total RNA and made 10x fold dilutions. I then added mRNA from a gene that I will not be looking at so that all samples had the same amount of total RNA before reverse transcription. To get the other mRNA I used in vitro transcription.
HTH
Cheers,
Anil.