The ability to monitor the real-time progress of PCR has completely revolutionized the way one approaches PCR-based quantification of DNA and RNA. The process of creating quantitative assays is streamlined because the construction and characterization of quantity standards are no longer required. Real-time quantitative PCR (qPCR) has made the quantification of DNA and RNA much more precise and reproducible because it relies on threshold cycle (Ct) values which are generated during the exponential phase of PCR; this is more accurate than quantification based on the PCR endpoint as used by earlier PCR quantification methods. The more gene copies present at the beginning of the reaction, the fewer number of cycles it takes to reach a point in which the fluorescent signal is first recorded as statistically significant (i.e. above background); it is this cycle number which is the Ct value.
The MiniOpticon qPCR detection system includes a compact, 2-color real-time detector built on the 48-well MJ Mini cycler. It is the smallest and most portable (18 x 32 x 33 cm, 6.8Kg) systems available for qPCR applications and gel-free PCR analysis. Its 2-color multiplexing capability allows for detection of a range of fluorophores (FAM, SYBR® Green I, TET and VIC). The MiniOpticon uses Peltier-based thermal control to produce quick ramping and accurate temperature for fast, reproducible runs.
The MiniOpticon system also includes Opticon Monitor software, a powerful yet easy-to-use package for setting up experiments and analysing results. In addition to absolute quantification, the software includes modules for relative gene expression analysis and genotyping. Melt curve analysis is also available, so you can confirm specificity without having to run a gel.
The MiniOpticon system uses illumination technology similar to that of the DNA Engine Opticon 2 system — samples are sequentially illuminated by a fixed array of 48 blue-green light-emitting diodes (LEDs). Each LED beam is precisely focused onto its corresponding well to ensure minimal crosstalk and light scattering. Crosstalk is further reduced by sequentially detecting each well. The LEDs efficiently excite fluorescent dyes with absorption spectra in the 470-505 nm rage. Emitted fluorescence is detected by one of two sensitive, filtered photodiodes (523-543 nm and 540-700 nm). This innovative no-moving-parts design allows accurate detection in a compact and robust package.
I use the MiniOpticon to determine the gene expression levels at different time points in cultured skeletal muscle cells treated with different concentrations of TNF-alpha. I used iScript™ One-Step RT-PCR kit with SYBR® Green I (Bio-Rad) to quantfy the mRNA transcription profiles of several genes of interest. Using the Opticon Monitor software allows me to quantify the expression level of genes of interest either by absolute or relative quantification.
Quantification using SYBR Green I Dye: The MiniOpticon real-time qPCR detection system can be used to accurately quantify the initial concentration of a target template in solution. In a quantitative PCR experiment, samples of known concentration are run, then a standard curve is generated by graphing the logarithm of known quantities vs. the amplification cycle at which the measured fluorescence for each sample exceeds the background (CT). Quantification of unknowns is accomplished by interpolating the CT for each unknown against the standard curve generated from the known samples. In this way, the initial concentration of the unknowns can be calculated to a high degree of accuracy. The large linear dynamic range over which the MiniOpticon can detect and quantify samples allows the testing of a wide variety of concentrations in a single experiment. This characteristic greatly reduces the need to perform time-consuming dilutions and multiple PCR experiments with individual unknown samples.
Multiplex Applications: The MiniOpticon real-time qPCR detection system can be used for multiplex qPCR which involves amplification of more than one DNA target in a single reaction tube. One obvious advantage to multiplexing is that it maximizes throughput and provides maximum information from each sample. It also provides greater confidence in results when quantifying and comparing different genes (e.g., gene of interest and housekeeping gene) in applications such as detection of genetically modified organisms (GMOs) and assessing relative gene expression. Another example of the power of multiplexing is in the unambiguous assignment of single nucleotide polymorphisms (SNPs) that can be achieved when probes for both wild-type and mutant sequences are present in the same reaction mixture.