Comparison of two separate real times - (Nov/03/2005 )
Hi,
I got a question for anyone out there....
I've got real time data from MCF-7 and MDA-MB-231 cells. The problem is.... i want to compare the amount of the gene present in each of these cell lines. is it possible to comapre the amount of RNA from a gene in the two cell lines, when they've been through two separate real times?
sorry, if i'm not being clear, not really clear on this myself.
the MDA-MB-231 cells are supposibly negative for my gene of interest. but i ran real time, and there's some lovely data on it. now, i know MCF-7 are positive for my gif (lots of lovely data), but.... if the MDA-MB-231 cells are really negative.... how do i compare the amount of gif RNA from each of these cell lines? to potentially show that in 231s the RNA is much less than in MCF-7.... potentially.
my PI wants to know why there's RNA for the gif, if the 231 are really negative. i'd like to know too...personally, i think's it's sensitivity of real time vs other methods. any other ideas?
help me please.
a million thanks,
vetticus
ps... the reason why MD231 cells are negative is through northern blot analysis. but looking back at the papers.... when MCF-7 cells aren't treated, they look negative too. anyone got a paper/reference saying that real time is more sensitive than northern blots?
Vetticus,
Are you sure that your RNA is DNA free (have you DNased it)? If not, this may account for your positive result in the 231. If you are very concerned, perhaps design a new set of primers where one spans two exons if possible so any gDNA in your sample would not be amplified, or at least be very distinguishable from your cDNA. As for the rest, if you are expressing your GOI as change in relative expression (or relative expression of one cell type versus another), I think you could do it if you used the same housekeeping gene. I would personally take a look at my data, then decide if they physically should be run together to decrease variability and increase my confidence. Good luck!
LabGirl
P.S. BioRad has some great customer service for these types of questions. If you are still unsure, you might contact them 
I got a question for anyone out there....
I've got real time data from MCF-7 and MDA-MB-231 cells. The problem is.... i want to compare the amount of the gene present in each of these cell lines. is it possible to comapre the amount of RNA from a gene in the two cell lines, when they've been through two separate real times?
sorry, if i'm not being clear, not really clear on this myself.
the MDA-MB-231 cells are supposibly negative for my gene of interest. but i ran real time, and there's some lovely data on it. now, i know MCF-7 are positive for my gif (lots of lovely data), but.... if the MDA-MB-231 cells are really negative.... how do i compare the amount of gif RNA from each of these cell lines? to potentially show that in 231s the RNA is much less than in MCF-7.... potentially.
my PI wants to know why there's RNA for the gif, if the 231 are really negative. i'd like to know too...personally, i think's it's sensitivity of real time vs other methods. any other ideas?
help me please.
a million thanks,
vetticus
ps... the reason why MD231 cells are negative is through northern blot analysis. but looking back at the papers.... when MCF-7 cells aren't treated, they look negative too. anyone got a paper/reference saying that real time is more sensitive than northern blots?
i ran both of the samples together, and....well the gof RNA from the negative cell line was almost non existant . basically, the real time was just a *lot* more sensitive than a northern blot.
Glad you got it worked out!
. basically, the real time was just a *lot* more sensitive than a northern blot.