John M. S. Bartlett.pdf - Bio-Nica.info

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after the PCR. This reduces the occurrence of contamination of subsequent assays
by previous PCR products, which is an important consideration for any PCR system.
A disadvantage of this system is that it may not be suitable for all target proteins.
We have found that for some target RNAs trial of a large number of primer sets may be
necessary before suitable primers are found. Also, the short amplicon length leads to an
increased chance of homologous products amplified compared with PCR using primers
for longer amplicons. We have found that the strict conditions may result in it not being
possible to find suitable primers/probe for some RNA species. In our experience, these
have been coagulation proteins, which are known to have a high degree of homology
with other proteins.
3. The above protocol will generate results as the cycle at which a set fluorescence threshold
(Ct) is reached for each standard/unknown for both t-PA mRNA (FAM) and 18S rRNA
(VIC). The standards are used to ensure that the difference between the Ct of the test RNA
species and the Ct of the 18S rRNA internal control (∆Ct) remains constant across all
mRNA dilutions in the range chosen for the standards (10 to 0.156× above). We can ensure
that this is the case by plotting the ∆Ct for each of the standards against the log total RNA
for each of the standards. If the gradient of the slope of this line is less than ± 0.1, this
indicates that the ∆Ct is remaining constant and that the “∆∆Ct∀ method can be used
to quantitate the mRNA for the unknowns as follows. The relative mRNA quantity of
various unknowns compared with a nominal baseline unknown are calculated by using
the formula 2 –∆∆ct , where ∆∆Ct is the difference in the ∆Ct of the test sample and the ∆Ct
of the nominal baseline sample.
The use of the ∆∆Ct method is advantageous when a large number of repeated samples
have to be analyzed because it removes the need for running a set of standards each time,
which is both time-consuming and increases costs. However, for many purposes, it seems
sensible to generate a standard curve each time to ensure the validity of the results.
References
1. Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Real time quantitative
PCR. Genome Res. 6, 986–994.
2. Desjardin, L. E., Chen, Y., Perkins, M. D., Teixeira, L., Cave, M. D., and Eisenach, K. D.
(1998) Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification
of IS6110 DNA in sputum during treatment of tuberculosis. J. Clin. Microbiol. 36,
1964–1968.
3. Goidin, D., Mamessier, A., Staquet, M. J., Schmitt, D., and Berthier-Vergnes, O. (2001)
Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and betaactin
genes as internal standard for quantitative comparison of mRNA levels in invasive and
noninvasive human melanoma cell subpopulations. Anal. Biochem. 295, 17–21.