The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful method to study the presence of a distinct RNA target sequence in a sample. It consists of two steps
i) the conversion of the RNA template to cDNA via a reverse transcriptase enzyme, and
ii) the amplification of the newly synthesized cDNA via PCR. In qPCR the amplification of DNA is monitored during cycling in real-time by fluorescence detection thus allowing conclusions about the initial amount of template in the sample.
Using probes labelled with different fluorophores enables multiplexing. In multiplex RT-qPCR more than one target sequence (e.g. a target gene and a control gene) are amplified in the same reaction. Therefore, every reaction can be quality controlled, which is of great importance in molecular diagnostics. Moreover, by using multiplexing reagent costs can be reduced and sample throughput increased at the same time.
RT-qPCR can be performed using two approaches called two-step and one-step. In the two-step approach, cDNA synthesis and PCR amplification are carried out in separate tubes. Two-step RT-qPCR is most often used in research, where greater flexibility, lower reagent costs and the setting up of cDNA archives are of concern. In one-step RT-qPCR both cDNA synthesis and PCR amplification are carried out in the same tube. This greatly simplifies the process and reduces handling errors as well as the risk of contamination. Therefore, the one-step RT-qPCR approach is most widely used in diagnostic applications.
Advanced Optical Scanner
Digital Pathology Software
The proximity ligation technology (PLA) is based on two primary antibodies targeting the protein(s) of interest. These antibodies will be recognized by a pair of secondary antibodies conjugated to oligonucleotides, which can form a DNA circle when bound in close proximity. After enzymatic activation and ligation, the DNA is amplified by rolling circle amplification. Then, the amplified DNA is detected by fluorophore-labelled detection probes, which hybridize to the complementary sequence. The reaction products can be visualized as distinct spots under a fluorescent or confocal microscope.