Junk/dimers on left of single product peak - (Sep/06/2006 )
I got a serious problem with fluctuation/noise/junk on left of single product peak when ran the cDNA (1000ng/ul) with different concentration of primers (chequered board, pmol/ul) i.e. (forw/rev) 50/50, 50/25, 50/10, 50/05, 50/1, 25/50, 25/25,..........1/50, 1/25, 1/10, 1/05, 1/1. The product Tm is 84 and whereas the forw primer Tm=59 and reve primer Tm=59. The product size is 105bp. I have tried using different annealing temperatures (55 & 56 respectively), but the problem of junk is consistent. Unfortunately am getting amplification in RT neg & NTCs at around 30-33 Ct. I have changed my reagents, water, tips and pipettes. Thinking whether the stock primers were contimated, I have ordered fresh primers and made the working concentration in a laminar hood.
Am using Stratagene's MX3005p machine and real time SYBR green-I mastermix kit. Nuclease free water from promega. For total RNA and DNase digestion, using Qiagens kit whereas for cDNA synthesis am using Superscript III RT (invitrogen).
I am in need of help because I have no one in my lab who knows about qPCR. It will be appreciated if someone help me in this. Please find the attached powerpoint slides of my run.
OK...here's the disclaimer: I do quite a bit of qPCR, but I would not consider myself an expert, allright? however, I can tell you what I think and how I would interpret these curves
for some of those (the spiky low peaks) I am guessing they are your NTC's and water-controls? they are fine; look at the scale. all the little spiky peaks are less than 3. this is not a significant problem
as for the 3 - I am assuming they are amplification melting curves? - that have a high peak on the right and the noise on the left - I think that noise is 'acceptable' if your data is reproducible and the peak is higher than 20 or so. for only one of these curves is the main peak high enough. I am guessing some non-specific binding is occuring, at low enough level that you can see the gist of your expression changes but that your data may not be tight enough to yield good statistics from one run or sample to the next.
OK, how to fix it: increase specificity? perhaps try a new set of primers; make sure your RNA is good (if there's degradation it can cause some of the noise you're seeing); check stuff like that?
Thanks for your suggestions. I have check the quality of RNA (260/280 >2.0) which I think is good. I have set up a run at high annealing temp i.e., 58. I hope that the small spiky peaks on right of product melting peak will disappear. If it doesnt work either, then I will try designing the new primers.
just a sec
how much higher than 2.0?