Real-Time PCR has become the “gold-standard” of DNA amplification reactions, allowing fast and accurate nucleic acid quantitation. The operation of the ABI Prism 7000 Detection system is similar to other Real-Time thermal cyclers, incorporating a fluorescent DNA binding agent into amplified DNA template thus, allowing accurate readings to be taken at defined intervals.
I have used the ABI Prism 7000 in conjunction with the Primer Express Software program, the SyBr Green PCR Master Mix, and the ABI Prism 7000 SDS Software (comes standard with the ABI Prism 7000) to quantify differences in gene expression from various cell and tissue samples. The Primer Express Software allows for the design of specific Real-Time PCR primers and probes, and is compatible with the default ABI Prism 7000 PCR program. Other lab members have used this system to test for differences in gene-copy number (genomic DNA) from different cell lines and to normalize levels of plasmid in transfected cell lines. The system can also be easily utilized for the construction and use of Taq-Man probes (a fluorescent probe that binds within the primer amplicon), but whether the increase in signal specificity justifies the cost of these probes must be determined by the individual researcher.
The experimental design and implementation are straight-forward. PCR-primer design using the parameters in Primer Express are optimized for the default reaction conditions of the ABI Prism 7000 thermal cycler (amplicon is generally 70-130 base pairs). To date, I have designed many primer-pairs using the default parameters and all have worked successfully. A reaction run is established using nanogram quantities of reverse-transcribed cDNA (less starting template can potentially be used), 8l of SyBr Green PCR Master Mix, and 10-20ng of each primer. To avoid contamination, I always remove genomic DNA using Promega’s “DNAse1” before reverse-transcription of the cDNA.
It is important to perform each PCR reaction in duplicate/triplicate for each sample to compensate for potential pipetting errors. This also enables calculation of standard deviations and means of expression. During amplification, the SyBr green (which is only weakly fluorescent when unbound) is incorporated into the dsDNA of the amplicons, where fluorescence is dramatically increased. Excitation and resulting emission spectra are detected by the filter sets in the ABI Prism 7000, resulting in a detailed set of fluorescence data for each sample. The level of SyBr green fluorescence is directly proportional to the amount of DNA present in each tube. At the conclusion of the run, the data for each sample is displayed as a typical PCR curve (including linear phase of amplification and end “plateau-phase”).
Using the software provided, a “threshold” can be set, allowing analysis of all linear phase samples. The read-out is presented as the cycle number where the linear phase for each sample crosses the threshold level. The rate at which the cycle number reaches the established threshold determines the quantity of DNA present in each sample (earlier = more DNA). For my studies, I always use a housekeeping gene such as GAPDH or -actin to normalize input levels between samples.
In summary, the ABI Prism 7000 Sequence Detection System is a fantastic system for accurately quantifying nucleic acids between different tissues, cell lines, or experimental treatments. High reproducibility, ease of experimental design, and accurate data collection/display allow for quick and rigorous data analysis. Although the cost may be prohibitive, the ABI Prism-SyBr green Real-Time PCR system provides a precise way to quantify nucleic acid expression.
Dworkin Sebastian
Graduate Student
Peter MacCallum Cancer Centre
Melbourne, Australia