RT-PCR Vs qRT-PCR help please! - (Jan/19/2008 )
Hi,
I was wondering if any one could help me, I'm going to be studying the expression of a couple of genes, in normal cells and then treated cells. I've only ever done it using RT-PCR never quantitativly.... so I was wondering should I do a regular RT-PCR first to see if there is any difference and then do the quantitative qRT-PCR? or do people just generally go straight to the real time?
I'm looking at five genes and having trouble designing real time primers so thought regular RT-PCR would be easier and get me started quicker... but then maybe doing regular RT-PCR would be wasting time and money 
(on primers etc..)
Any help would be great!
Thanks!!!
I was wondering if any one could help me, I'm going to be studying the expression of a couple of genes, in normal cells and then treated cells. I've only ever done it using RT-PCR never quantitativly.... so I was wondering should I do a regular RT-PCR first to see if there is any difference and then do the quantitative qRT-PCR? or do people just generally go straight to the real time?
I'm looking at five genes and having trouble designing real time primers so thought regular RT-PCR would be easier and get me started quicker... but then maybe doing regular RT-PCR would be wasting time and money
(on primers etc..)Any help would be great!
Thanks!!!
In my experience I would..
Just do RT-PCR (semi-quant.) if I wanted to see a result. ie: to see if there were massive differences in expression levels. Cheaper and less hassle (in terms of designing primers).
But if it were for a paper, then qRT-PCR. It's expected in good journals.
To me, qRT-PCR is something you use to get some fancy numbers when you could not see any difference in band density on a agarose gel. Probably for high throughput screening, it has its advantage of saving time over regular PCR.
Thanks for that!! It is to be published eventually... (Just Started my PhD) And I'm trying to plan things out. So if I am going to do the qRT-PCR is there any need to do a RT-PCR first? or am I adding an extra step that isnt needed??
Thanks a million guys!
I'm sorry to partially disagree. My opinion is that the usefulness of RT-PCR in detecting changes in gene expression will depend on the experimental design, that is, if you are expecting a huge difference in expression, RT-PCR will probably do, but if you're looking at changes in a more physiological range, then the sensitivity limit for RT-PCR won't allow you to see them, as if I'm not wrong, the dinamic range is 10-fold for RT-PCR compared to 2-fold for qPCR. As a side note, it's always tricky to optimize the first sets of primers for qPCR, but once you get the trick, it's really quick!
I apologize if I'm wrong!