is cDNA concentration 200 ng/ul too high for qRT-PCR-Rea

The gene we are interested in looking at is not very abundant. At 100 ng/ul, the CT value is 30.9In 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? -PRK-

QUOTE(PRK @ Mar 9 2006, 11:37 AM) [snapback]43694[/snapback] 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? Ever heard about primer dimers? -micky_74-

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?

-aimikins-

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

-PRK-

QUOTE(PRK @ Mar 9 2006, 11:52 AM) [snapback]43700[/snapback] Of course, I have "heard" of primer dimer. Not sure how to determine if it is a primer dimer artifiact or mispriming. What is the difference between primer dimer artifact and mispriming? -micky_74-

prk, have you run a gel?

-aimikins-

QUOTE(aimikins @ Mar 9 2006, 12:30 PM) [snapback]43704[/snapback] prk, have you run a gel? Yes, if you have a good pcr you can see your products, if it is wrong you can see primer dimers and your products, that is what I can see on my gels -micky_74-

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 � 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.

-soluene-

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.

-PRK-

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

-PRK-

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?

-aimikins-

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 �/� 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-

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?

-PRK-

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?

-soluene-

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  

-aimikins-

QUOTE(soluene @ Mar 9 2006, 04:43 PM) [snapback]43717[/snapback] 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. 900nM seems high even we have used AB Taqman gene expression at 250nM and has worked fine...we feel 900nM is too high -BusyBee-

QUOTE 900nM seems high even we have used AB Taqman gene expression at 250nM and has worked fine...we feel 900nM is too high 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. -soluene-

QUOTE(soluene @ Mar 10 2006, 05:44 PM) [snapback]43852[/snapback] QUOTE 900nM seems high even we have used AB Taqman gene expression at 250nM and has worked fine...we feel 900nM is too high 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. looking forward to hear about your results from 0.5x -BusyBee-

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