For general laboratory use. Others LightCycler RNA Master HybProbe GLU LightCycler® RNA Master HybProbe 3.5.5.1.2.6 03018954001 LC RNA Master Hybridization Probes LightCycler RNA Master HybProbe 04038377017198 Reagents, kits 1 kit 96 reactions of 20 μl final volume each false The LightCycler® RNA Amplification Kit HybProbe is designed specifically for the HybProbe detection format using the LightCycler® Carousel-Based System. The kit provides reagents, including RT-PCR enzyme mix, reaction mix, MgCl2, and Water, PCR Grade for very sensitive detection and quantification of defined RNA sequences using the LightCycler® System (suitable RT-PCR primers and HybProbe probes must be supplied).The kit is especially suitable for difficult RNA populations, as the elevated incubation temperature during the reverse transcription step will help to overcome secondary structures. The hot start feature will minimize mispriming during the initial phase of the reaction and therefore overall sensitivity of RT-PCR is increased. It can also be used to genotype single nucleotide polymorphisms (SNPs) and analyze mutations.In addition, the kit can be used with Uracil DNA Glycosylase, heat-labile to prevent carryover contamination during PCR. en Easy-to-use Reaction Mix for One-Step RT-PCR, using HybProbe probes with the LightCycler® Carousel-Based System. en The hot start feature of the LightCycler® RNA Amplification Kit HybProbe is achieved by using Tth DNA Polymerase, in combination with aptamers. Tth DNA Polymerase is a thermostable enzyme with RNA-dependent reverse transcriptase activity and DNA-dependent polymerase activity, allowing the combination of RT and PCR in a single-tube reaction. Aptamers are dedicated oligonucleotides that bind in the active center of the polymerase and prevent attachment to nucleic acid targets at temperatures below the optimal reaction temperature of the Tth enzyme. Therefore, no primer elongation occurs during setup of the reaction and the following heating phase prior to the RT step. At higher temperatures, the aptamers are released from the enzyme and RT or DNA polymerization can be initiated. In addition, the recommended incubation temperature for reverse transcription with Tth (+61°C) is helpful to overcome secondary structures of RNA. This results in highly specific and efficient cDNA synthesis that leads to highly specific and sensitive PCR. Hot start with aptamers is highly effective and very convenient because it does not require additional incubation steps, pipetting steps, or an extension of reaction time. The hot start protocol with aptamers does not interfere with other enzymatic processes, the online detection of amplification products, or subsequent handling steps.HybProbe probes consist of two different short oligonucleotides that bind to an internal sequence of the amplified fragment during the annealing phase of the amplification cycle.The basic steps of DNA detection by HybProbe probes during real-time PCR on the LightCycler® Carousel-Based System are:The donor dye probe has a fluorescein label at its 3' end and the acceptor dye probe has a red fluorophore label (LightCycler® Red 610*, LightCycler® Red 640*, Cy5, or Cy5.5) at its 5' end (it is 3'-phosphorylated, so it cannot be extended). Hybridization does not occur during the denaturation phase of PCR. As the distance between the unbound dyes prevents energy transfer, no fluorescence will be detected from the red acceptor dye during this phase.During the annealing phase, the probes hybridize to the amplified DNA fragment in a head-to-tail arrangement, thereby bringing the two fluorescent dyes close to each other. Fluorescein is excited by the light source of the LightCycler® Carousel-Based System which causes it to emit green fluorescent light. The emitted energy excites the red fluorophore by fluorescence resonance energy transfer (FRET). The red fluorescence emitted by the acceptor dye is measured at the end of each annealing step, when the fluorescence intensity is greatest.After annealing, an increase in temperature leads to elongation and displacement of the probes.At the end of the elongation step, the PCR product is double-stranded, while the displaced HybProbe probes are back in solution and too far apart to allow FRET to occur.HybProbe probes that carry different red fluorophore labels can be used separately (for single-color detection experiments) or combined (for dual- or multiple-color detection experiments). Color compensation is not necessary for single-color detection experiments. However, if you are using HybProbe probes to perform dual- or multiple-color experiments in a single capillary, you must also use a color compensation file. Color compensation may be applied either during or after a run on the LightCycler® Carousel-Based System.See the LightCycler® Operator's Manual and the Instructions for Use of the LightCycler® Color Compensation Set for more information on the generation and use of a color compensation file, or key.LightCycler® Red 610 and Cy5 can only be used on a LightCycler® 2.0 Instrument.How this Product WorksLightCycler® RNA Master HybProbe is an easy-to-use hot start reaction mix, specifically adapted for one-step RT-PCR in 20 μl glass capillaries using the detection format of the HybProbe probe on the LightCycler® Carousel-Based System. Amplification and online monitoring of the template RNA is achieved by a combined procedure on the LightCycler® Carousel-Based System Instrument. The results are interpreted directly after completing the PCR and Melting Curve. The amplicon is detected by fluorescence using target-specific HybProbe probes (not supplied with the kit).The LightCycler® RNA Master HybProbe provides convenience, high performance, reproducibility, and minimizes contamination risk. All you need to supply is the template RNA, RT-PCR primers, and HybProbe probes.In principle, the LightCycler® RNA Master HybProbe can be used for the amplification and detection of any RNA target. However, you would need to adapt each amplification protocol to the reaction conditions of the LightCycler® Carousel-Based System and design specific RT-PCR primers and HybProbe probes for each target. Refer to the LightCycler® Operator's Manual for recommendations.The amplicon size should not exceed 750 bp in length. For optimal results, select a product length of 500 bp or less.The performance of the kit described in this Instructions for Use is guaranteed only when it is used with the LightCycler® Carousel-Based System. en