Methods and Applications Guide Introduction to Quantitative PCR
Document source : www.chem.agilent.com
24
Running a reference dye can still have some value, because in some
cases it can result in somewhat cleaner looking data. Also, even if
normalization is not performed, it can be very useful in the
troubleshooting process if you see any unexpected results in the
amplification plots of your reporter dyes. If signal is particularly low
or high, or if there is an odd shift in the fluorescence level, you can
check to see if similar effects are seen in the reference dye profile,
which should normally run flat. Being able to determine whether
effects are seen only with your reporter dye or with both the reporter
and reference dyes can often allow you to discriminate probe problems
from potential instrument problems.
If ROX is used as a reference dye, from an instrument standpoint
there is a difference in the concentration of reference dye that should
be used in the Mx instrument vs. some other systems. The white light
excitation in the Mx system and the system's dye- specific filters will
excite and measure the fluorescence for ROX very efficiently. We
designed the ROX filer set to be very sensitive so this dye channel can
be used for actually detecting fluorophores labeled with ROX or Texas
Red. In systems that do not allow excitation at ~584 nm (including
laser- based systems), ROX is excited very inefficiently, so a higher
concentration of the reference dye is used to compensate for the low
ROX signal. If a kit that is designed for one of these systems is used
in the Mx system, the high concentration of ROX will create
oversaturated signal on the ROX channel and result in the normalized
data containing more noise than the non- normalized data. In the Mx
system, ROX should be used at a final concentration of approximately
30 nM of free dye. If a master mix containing a final ROX
concentration closer to 300 nM is used, it is recommended that the
non- normalized baseline- corrected amplification plots (dR) be used for
analysis rather than the normalized baseline- corrected plots (dRn).
Some master mixes contain a short oligonucleotide labeled with FAM
and ROX that causes emission from ROX by energy transfer or FRET.
The presence of these oligonucleotides is compatible with fluorescence
detection in the Mx system and should not cause any difficulty in
normalization.
Summary :
In systems that do not allow excitation at ~584 nm (including laser- based systems), ROX is excited very inefficiently, so a higher concentration of the reference dye is used to compensate for the low ROX signal. If a kit that is designed for one of these systems is used in the Mx system, the high concentration of ROX will create oversaturated signal on the ROX channel and result in the normalized data containing more noise than the non- normalized data.
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