doubts about melting curve, Ct... - understanding results from 1st real time PCR (Sep/03/2008 )

HI guys,
I've just run my first real time PCr and now I have to face the results:

I'm using two step reaction with Sybr-Greener on a rotorgene 3000.
this first run was to check amplification efficiency, primer dimers, gDNA contamination using 18s primers.
I made 4-points serial diluitions starting with 100 ng/ul RNA (in triplicates), run a NTC in duplicates, NRT ctr for 3 samples (100 ng/ul RNA DNase treated). 2 um template in a final volume of 20 ul, 1 ul primers (10 uM)
cycling conditions:
50°C 2'
95°C 2'
40x: 95°C 15", 54°C 30", 72°C 30"
melting step.

that's what I got:
undiluted standard: Ct=7,5
1:10 dil: Ct= 8,3
1:100 dil Ct=10,5
1:1000 dil Ct=14
NTC: Ct= 24,9
NRT sample1: Ct= 20
NRT sample2: Ct= 21
NRT sample3: Ct= 18

Now I'm wondering why
1) the Cts are so low (at least in the serial dilutions)? Too much template?
2) should I worry about the amplification occurring in the NTC and NRT, or the Cts are high enough to be safe from contaminations?
3) when I analyze the melting curve:
undiluted standard: MT= 89°C
1:10 dil: MT= 88,5°C
1:100 dil MT=88,5°C
1:1000 dil no peaks = no amplification? to high diluition?

but the weird thing is that the NTC has a MT=82°C, the 2nd replicate has MT=81,5°C
I was expecting to see something around 60°C that's the MT of my primers...
2nd weird thing:
NRT sample1: MT= 87,5 and 86,8
NRT sample2: MT= 87,2 and 86,8
NRT sample3: MT= 87,3 and 87.
if there is gDNA contamination, why is the MT different from the amplified cDNA one?
I can't see (or recognize) peaks due to primer dimers, but when I run the samples on a gel, I've got primer dimers....

And last questions:
is it ok using 3 steps during the cycling (95°C 15", 54°C 30", 72°C 30"), or should I use two steps like 95°C 15", 54°C 1'?

Sorry for all these questions, every hint is very welcome!!
thanks
Raffaella

Hi

You write you use a two step reaction, so your reverse transcription reaction (step 1) takes place in a different tube than your real-time PCR reaction (step 2)? Is that right? I just got confused because you wrote you made the serial dilution starting with 100 ng/µl RNA. That sounded like a one step reaction, means reverse transcription and PCR in one tube.
Anyway, in my two step set-up I use only 50 ng/µl of total RNA in the reverse transcription reaction. So you might use too much template in your real-time PCR, especially if you use only mRNA. So your early Cts values might probably be explained by this.
I would worry about your NRT samples. They look like there is contamination with genomic DNA. To my experience DNA digestion does not work very well. I Use DNase and spin columns and still find genomic DNA in my samples. So to avoid amplification of gDNA it’s best to work with intron spanning primers.
Your NTC is also a bit early, might be a contamination too. Do you spatial split mixing the PCR-mastermix and adding the template? It’s always good to prepare your mastermix in a DNA-free room. Your high melting temperature for in your NTC also points in this direction.
Third I wonder about your 1:1000 dilution. You got a Ct of 14 but no product in the melting curve? I don’t think this dilution is too high. I used 1:729 dilution step in a three-times dilution series and it worked quite well. Remember your cDNA content is the double of mine if our RTs work more or less the same.

Good luck!

Jan

First thing you have to confirm you did not have contamination in your blank (NTC). Secondly, make more dilution for your template, since the ct for the diluated samples you were using is too small. Optimally Ct is greater than 15 but less than 30. So you can propose how much dilution you will start based on the results you got and 2-fold dilution give 1 deltaCt.

Based on the results you gave, it seemed your NTC was contaminated.

I always get positive reactions in my NTC with 18s. I think it may be carry over DNA from the recombinant Taq. However, it normally only comes up >cycle 28 and my sample come up between cycles 8-11, so I'm not very worried about it.

thanks for all your comments...

Jan you are right, it wasn't clear. I'm using a 2 step reaction and I reverse transcribed 100 ng of total RNA. I repeated the run with only 1 ul of template cDNA instead of 2ul (that's how much I used in the first run) and the Ct now it's around 13 for the undiluted cDNA, and Ct=17 for the 1:10 dilutions. and there has been an improvement in the NTC Ct as well, it's 25 with the 18s primers, and 34 for the other set of primers I'm using. is the ct of the 18s NTC far enough to considere the eventual gDNA contamination not interfering with my analysis? and actually with all primers I'm using the NTC Ct is much more farther than the NTC CT with 18s, so maybe I'm having something similar to what happens to Maset

and the NRT Ct are around 24-25... could it be that for this 2nd run I used a robot to dispense mastermix and template?

I still have to optimize my assay but step by step I'll get there, thanks to all your suggestions too. next point is to increase the efficiency. I had a look around but I couldn't find any practical advices on [Mg²⁺] gradient. Do you have any?

Thanks again,

Raffaella

Hi,

I can quote only my Quantace protocol. They say 3mM MgCl² is sufficient for most reactions. To optimize the reaction they recomend a series from 3.0 mM to 7.0 mM MgCl² per reaction in 0.5 mM steps (3.0; 3.5, ...).
Maybe it helps...
Jan