SD of individuals within groups - (Oct/08/2006 )
Hi all, i´ve been dealing with the RealTime for a couple of months. After getting my primers working for my species, beautifully, i run some Real Time reactions but my results are kind of..... unexpected..
I have 4 normal and 4 experimental animals. I run a standard curve (one dilution, as ive been told is enough if its accurate) wich shows an excellent R2 and efficiency above 0.9. The Cts of my samples, however, span from 22-25 within the same groups, and arent very different between groups. (so i would say, theres no difference between groups).
All tissue was extracted the same way, within two days, stored in RNAlater, extracted with the same kit, the quality of RNA seemes to be very good accorgind to 260/280 absorbance, and and retrotranscribed in the same reaction (1 microgram of tissue)..i mean, ive done everything i could to normalise them, but the Cts fluctuate... Which should be the normal Ct range within samples of the same groups to be acceptable??
I tried to use GAPDH but didnt´t succeed. Beta-actin, but didnt amplify in RealTime (and as well, id expect it to change with the experimental samples as i do regeneracion of optic axons...).
For 18S, i couldnt use the cDNA cause i retrotranscribed with OligodTs. Do you think using a mixture of oligodTs and hexamers for 18S could be a good option?
Plz, feel free to comment on ANY of the procedures i described, as it may be helpful to me.
Thanks in advance!!
First off I'd say you have most things covered. I would say that n=4 per group is perhapes a little small, we would typically use a minimum of 6 animals per group if not more, this will improve the data. When you say the Cts aren't very different , between groups, is this for your target gene, if so have you looked at the delta Ct? Are you running each sample in triplicate?
As an aside, since you are looking at regeneration, have you fully validated a reference gene, as you mention beta-actin would be expected to cahnge but so would most others, have you thought of using a glial marker that perhapes is unchanged (just a thought not really my area)?
hope some of this is helpful