is cDNA concentration 200 ng/ul too high for qRT-PCR - (Mar/09/2006 )

The gene we are interested in looking at is not very abundant. At 100 ng/ul, the CT value is 30.9
In the literature that I have looked at, people seems to using much lower levels of cDNA. I would actually like to increase the concentration of cDNA to 200 ng/ul but am wondering if this can cause any problems.
I use SYBR green.

Also, my water blank with reference gene(18S) has a CT value of 32.0. I have run this with DEPC water, and two different bottles of sterile MQ water with the same result. What is this?

what do your melting curves show you for your blanks? usually it's random garbage, really tiny, and nothing that will affect your results...make sure there isn't anything that looks remotely like a peak...but the fact that your Ct is only 30.9 is not so cool, it makes it barely distinguishable above background for the machine

so, tell me about your relative efficiencies here. and, I'm sure you are probably all over this already, but how's your RNA? if you have poor template for whatever reason it would probably take more to get good amplification. how's your RT reaction? tell us more about your protocol

OH, hey, in my personal experience...once your Ct gets above 32 or so, all other things being equal, your statistics typically go to hell anyways upon repetition...I try to get my stuff in the 18-26 range before I consider it to be good reliable data with high reproducibility.

anyone else have thoughts on these high Ct's?

Of course, I have "heard" of primer dimer. Not sure how to determine if it is a primer dimer artifiact or mispriming.

prk, have you run a gel?

PRK, how are you measuring your cDNA concentration? Just in the last few weeks, I figured out that I had very exaggerated A260 readings due to phenol contamination in my RNA. So when I thought I had 45 µg of RNA, I really only had maybe..10? In addition, phenol inhibits PCR. In a 2002 publication by Agilent, they found that 0.5% (w/v) phenol can exaggerate true RNA readings by 300%. If you are interested in that ref, let me know - I can give it to you.

Aimikins, Thanks for your insights. I am going to run more water blanks with lower primer concentrations. There definitely are peaks in the blanks. And banding in the gel..
Currently, the primer is at 400uM. I am going to test at 100 um, 80, 60, and 40uM. If this is the problem, it will help with my effieciencies as well.

Soluene,
Does the reference help you determine if there is phenol remaining in the sample? I use a nanodrop to estimate my cDNA. It usually reads about 1200 ug/ul