Lightcycler RT-PCR












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            Supplier               ROCHE s.r.o.                             
            Year of acquisition    2018                                     
            Price                  0.6 milion. CZK                          
                                                                            
            Financing              OP VVV CORE FACILITIES [ URL "LFHKEN-192.html "]
                                   CZ.02.1.01/0.0/0.0/16_017/0002515        
            Responsible Person     prof. PharmDr. Emil Rudolf, Ph.D.        


            DNA carries genetic information and determines the development of the whole organism. The 
            of its structure and function was a major breakthrough and subsequently led to a major dev

            molecular biological analysis. This analysis is one of the most important components of di
            is widely used not only in basic and applied genetic research, but also in fields such as 
            zoology, botany, clinical diagnostics, and forensic science. From a medical point of view,

            biological analysis is mainly used in fields such as pharmacogenetics/genomics, nutrigenom
            preimplantation diagnostics, microbiology and other clinical fields, which generally inclu
            analysis.


            In our department we use cell cultures as an experimental model, which are nowadays an ind
            used in research. Among other things, they are used, for example, for the study of anti-ca

            targeted therapy. In the last two decades, their use in the study of tumor biology has pro
            for better understanding of tumor development, metastasis progression and interactions wit
            stroma. The use of cell lines has become an integral part of the study of molecular biolog

            characterization of genetic and epigenetic processes, and the study of prognostic and pred
            for possible individualization of therapy.



            The Lightcycler 96 is an instrument that allows quantitative determination of DNA quantity
            of gene expression by absolute or relative quantification of gene expression in real time 

            fluorescence measurements. It is a gradient real-time PCR instrument that uses Peltier cel
            to allow the rapid temperature changes required for this technique. In addition, high-reso
            (HRM) can be performed to allow detection of mutations in samples using melting temperatur

            with specifically designed probes covering individual mutations. This analysis allows the 
            differences between two DNA sequences under test down to the single nucleotide difference 
            single nucleotide polymorphism (SNP) detection or mutation analysis.


            The instrument is equipped with a 96-well block in which samples can be analyzed in indivi
            well strips or 96-well plates, allowing up to 96 samples to be tested simultaneously in a 

            This speeds up both sample preparation and stand-alone analysis, thereby increasing proces

            In our laboratory, we have been using a PCR cycler that had a built-in camera around which

            were moved to capture the fluorescence. In the Lightcycler96, the fluorescence of all samp
            simultaneously using a CCD camera that is pointed at each well separately, thus avoiding m
            camera and samples. This ensures that all measurements are compact and the fluorescence in

            measured under the same conditions.

            For fluorescence measurements during analysis, an LED is used as the excitation source and

            excitation and 4 emission (detection) filters can be used and combined on the instrument, 
            different fluorophores to be analysed simultaneously in one reaction. The Lightcycler96 ha
            excitation/emission options: 470 nm excitation/ 514 nm emission (e.g. for the detection of

            SYBR, FAM, ResoLight dye); 533 nm excitation/ 572 nm emission (e.g. for the detection of f
            SYBR, FAM, ResoLight dye); 533 nm excitation/ 572 nm emission (e.g. for the detection of f
            SYBR, FAM, ResoLight dye). VIC, Hex, Yellow555); excitation 577 nm/emission 620 nm (e.g. f

            fluorophores: Red610, Texas Red), excitation 645 nm/emission 697 nm (e.g. for detection of
            Cy5).


            Thanks to the purchase of the Lightcycler96 instrument, we have supplemented the instrumen
            molecular biology laboratory, which will help us to obtain information about cellular proc
            DNA level and also allow a more comprehensive assessment of all physiological and patholog

            which could in the future improve the effectiveness of treatment strategies for some serio
            instrument is used by students of the Faculty of Medicine and Pharmacy of Charles Universi
            their postgraduate studies and allows them to process biological samples more efficiently.

            results will be used by the students for publishing activities, representation of the facu
            and international conferences and last but not least for successful completion of their st


            Author: RNDr. Veronika Skarková, Ph.D.