4 replies to this topic

[Mohamed samir]

Dear all,
I just surprised as I got a crazy curve in the real time PCR run for miRNA detection. The late amplification is not a matter but what is annoying me actually, the fluctuated curve from its begining. Look at the attached picture and please give me explanation for this , shall I consider it or not ? is this normal ?

Best regards

[jerryshelly1]

What is the fluorescent incorporation dye you are using?  Sometimes non-PCR fluctuations can occur.  This is what you are seeing.  Did someone leave out the polymerase mixture at room temperature for to long?  I would try running a control sample with the same dye to see if you still see this fluctuation.  It could also be caused by bubble formation, heat causing your plate to warp, or your optical cover film may have had an oily smudge on top of the well.  A bunch of different things could have happened, I wouldn't necessarily disregard your data.

Edit: Do you run your samples in doublets?

Edited by jerryshelly1, 24 May 2013 - 09:07 AM.

[Mohamed samir]

SO thanks for giving me these fruitful things. I used SYBR green dye. Why do u think that leaving the mic at room temperature might cause this ? I left it for a while but in ice dose it matter ?
Also, what do do you mean by control sample ? are u meaning control positive standard gene? how my optical cover may carry oil smudge , I brought it from the store directly ? I run my sample in dublicate ( the technical replicate ) and in triplicate as a biological replicate

I would appreciate a detailed answer

[jerryshelly1]

Leaving your polymerase at room temperature and exposing it to light will have an affect the polymerases stability.  If you leave it in the light, it has the potential to photobleach your fluorescent pigment.  Leaving your fluorescent dye on ice should not be a problem.  I was referring to a control sample that you know shows good amplification in the past.  Preferably a sample that uses the same primers are your experimental sample.  Primer degradation could alter your Ct values.  If your primer begins to degrade, small nucleotide sequences can bind, replicate and give you small fluctuations (probably not your cause, you have minimal fluctuations).  Your samples are measured by their fluorescence.  If your plate is warped, it you have a bubble formations or a smudge on your optical film, this will hinder the machine to take an accurate reading.  This will cause small deviations in your fluorescence reading each round of replication.  Whenever I finish loading my plate, I spin it down and give the optical film a quick wipe.  Who knows if something inadvertently got on your optical film.

I was curious to see if you ran this sample in doublets because I wanted to know if this was a average of the two samples.  If this is not an average, did you other sample show this same fluctuation?

After you finish your qPCR, run your samples on a gel.  Are you still getting a sharp band from when you initially tested your primers and polymerase at the current cycling conditions.

In all honesty.  Your curve is good.  The relative fluctuations are probably just the result of the Sybr green being uncooperative.

[Mohamed samir]

Thanks, Actually this fluctuation is seen in one of the dublicate not all of them. However, you refere to degraded primer. how can we know that our primer start to be degraded ?