is cDNA concentration 200 ng/ul too high for qRT-PCR - (Mar/09/2006 )
hmmm
400 seems high, but there are many different ways to do it...
I have 6 genes that I routinely amplify; there are differences from gene to gene by my most optimal efficiencies run between 100 and 150
soluene, how much primer do you add?
PRK-
No, unfortunately the reference doesn't help with that. I did kind-of an extensive experiment using two quantitation methods that led me to believe I had phenol in my samples. I would be happy to explain it all if you would like.
I don't use nanodrop, but it looks as though it uses absorbance to quantitate nucleic acid. Phenol absorbs at 260 and may exaggerate your readings if it is present.
Also, this is not at all meant to insult you, but are you measuring purified cDNA on nanodrop? 1200 µg/µL seems incredibly high. I did not know this when I first started, but you cannot quantify cDNA after RT unless you purify it first. If you are doing that (measuring without purifying), you are also measuring all the RT components in your tube. This can also cause exaggerated concentrations of cDNA.
Aimikins-
I actually add 900 nM primers because I use pre-designed TaqMan Gene Expression assays. AB always recommends 900 nM primers whether it is their assay or your own. Please note these are TaqMan assays and not SYBR green. I think their recommendation for SYBR green is much lower but I don't use it so I'm not sure.
Soluene,
When I started this Real Time work about 6 weeks ago, I purified the cDNA first. But I had so little left after this (10-15 ng/ul), that I did not have enough cDNA to make the concentration that I needed. So then I started using the non-purified. Well crap..now what do I do?
Hmm...so it looks as though you were simply adding a lot less RNA to your PCR reactions than you thought you were, but I think that is ok.
As for what to do....I'm fairly new at RT PCR, too (5-6 months), so others may disagree with me on this...But here I go:
Using non-purified cDNA won't harm your real time PCR results (assuming there aren't many inhibitors, etc. in your cDNA and that your primer design is good). You just don't know how much are adding to your reactions. If you are using the ddCt method, and if you have enough cDNA to play with, I would just try making lower dilutions of your samples and see if you can get the Ct's within an acceptable range. The ddCT method is all relative anyway.
What do others think?
that looks OK to me?
the tricky part is, with the next batch of RNA much of the optimization will have to be repeated again when you can make a better guesstimate of how much is being added, for future work
I actually add 900 nM primers because I use pre-designed TaqMan Gene Expression assays. AB always recommends 900 nM primers whether it is their assay or your own. Please note these are TaqMan assays and not SYBR green. I think their recommendation for SYBR green is much lower but I don't use it so I'm not sure.
Hiya BusyBee-
Yeah, actually I agree with you! The pre-designed kits (includes fwd primer, rev primer, and probe in 1 tube) are supplied at a 20X concentration, and when you prepare the samples, 1X leaves you at 900/900/250 nM concentrations. I haven't had a chance to try it at 0.5X, but the problem is that if we don't use it at the recommended concentrations, AB can say "well, we can't guarantee it will work for you" (and of course, we wouldn't won't buy as much either!), and then our clients will say, "well, we only want things that are guaranteed"...You get the idea, I'm sure
. When I have time, I'll probably do internal studies to show that 0.5X is just as good as 1X. Hiya BusyBee-
Yeah, actually I agree with you! The pre-designed kits (includes fwd primer, rev primer, and probe in 1 tube) are supplied at a 20X concentration, and when you prepare the samples, 1X leaves you at 900/900/250 nM concentrations. I haven't had a chance to try it at 0.5X, but the problem is that if we don't use it at the recommended concentrations, AB can say "well, we can't guarantee it will work for you" (and of course, we wouldn't won't buy as much either!), and then our clients will say, "well, we only want things that are guaranteed"...You get the idea, I'm sure
. When I have time, I'll probably do internal studies to show that 0.5X is just as good as 1X.