QPCR result interpretation

[vikuba]

Hi All,

I am facing some difficulty in interpreting my results my Duplex qpcr. Since i am using laser capture microdissection to capture the cells , i usually work with low quantity of rna, the attached file is from Biorad Icycler software, the three samples are replicates. can you suggest me how and what would you interpret from this sort of data. Like, is it a failed reaction / or i have too little template / or I can take the Ct values ....... ? I would really appreciate your valuable suggestions in this regard. pleased find attached the zip file containing the screen shot from Icycler software. Thanks again

[danfive]

What kind of qPCR--SYBR, Taqman probe? Is this a one step RT-PCR?
For now this is a failed reaction.
You should also bring the baseline (x-axis) to intersect the y-axis at zero. The negative flourescence (in negative y axis) is not of any use in qPCR.

[vikuba]

Originally Posted by danfive

What kind of qPCR--SYBR, Taqman probe? Is this a one step RT-PCR?
For now this is a failed reaction.
You should also bring the baseline (x-axis) to intersect the y-axis at zero. The negative flourescence (in negative y axis) is not of any use in qPCR.

This is a one step rt pcr and it uses TAqman probe. Can you suggest me some of the possible causes of this, one of the reasons may be low template, but one of the target gene in the same plate worked pretty well while rest of the genes showed results like the one i presented. Also, some time I find that the the cycles start at very high values on Y axis and then they come down.Can you tell me why do they sometimes start from high values on y axis instead of a horizontal line.

[danfive]

Originally Posted by vikuba

This is a one step rt pcr and it uses TAqman probe. Can you suggest me some of the possible causes of this, one of the reasons may be low template, but one of the target gene in the same plate worked pretty well while rest of the genes showed results like the one i presented. Also, some time I find that the the cycles start at very high values on Y axis and then they come down.Can you tell me why do they sometimes start from high values on y axis instead of a horizontal line.

The very low template is most likely the problem. When extracting tiny amounts of RNA, you will inevitably lose some to tube/pipette tip walls. Also if the tissue is fixed, paraffin-embedded etc. the RNA/DNA is most likely sheared, and that includes some of your mRNA copies.
As to the high background/noise signal this can be reduced by careful pcr reaction prep, mostly preparing them on ice, and keeping the exposure to light minimal. Also you can let the reactions sit on the thermocycler (with lid closed, so it is in absolute darkness) for about 2 minutes before beginning the cycler.

What is the thermocycle profile for the RT-PCR?