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The amplicon is detected by fluorescence monitoring using a target-specific hydrolysis probe (not provided by the kit). Amplification and online monitoring of the template RNA is achieved by a combined procedure on the LightCycler® 480 or LightCycler® 96 Instrument. The results are interpreted directly after completing the RT-PCR.

The LightCycler® 480 RNA Master Hydrolysis Probes provides convenience, high performance, reproducibility, and minimizes contamination risk. Only template RNA, PCR primers, hydrolysis probe, and Mn(OAc)2, have to be added.

The hot start feature of the LightCycler® 480 RNA Master Hydrolysis Probes 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 reverse transcription 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 Tth DNA Polymerase. Therefore, no primer elongation occurs during setup of the reaction and the following heating phase prior to the reverse transcription step. At higher temperatures, the Aptamers are released from the enzyme, and reverse transcription or DNA polymerization can be initiated. In addition, the recommended incubation temperature for reverse transcription with Tth DNA Polymerase (61°C) is helpful to overcome se-condary structures of RNA. This results in highly specific and efficient cDNA synthesis, which 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.
Test Principle
Sequence-specific detection of PCR products relies on sequence-specific oligonucleotide probes that are coupled to fluorophores. These probes hybridize to their complementary sequence in target PCR products. Probe chemistries that are suitable for use in the LightCycler® 480 Instrument include single-labeled probes, hybridization probes, and hydrolysis probes. For the LightCycler® 96 Instrument use hydrolysis probes. Hybridization and hydrolysis probe chemistries use the so-called FRET principle. Fluorescence Resonance Energy Transfer (FRET) is based on the transfer of energy from one fluorophore (the donor or reproter) to another adjacent fluorophore (the acceptor or quencher).

Hydrolysis probe assays can technically be described as homogeneous 5'- nuclease assays, since a single 3'-non-extendable probe, which is cleaved during PCR amplification, is used to detect the accumulation of a specific target DNA sequence. This single probe contains two labels, a fluorescent reporter and a quencher, in close proximity to each other. When the probe is intact, the quencher dye is close enough to the reporter dye to suppress the reporter fluorescent signal (fluorescent quenching takes place via FRET). During PCR, the 5'-nuclease activity of the polymerase cleaves the hydrolysis probe, separating the reporter and quencher. The reporter dye is no longer quenched and emits a fluorescent signal when excited.

The LightCycler® 480 Instrument can detect hydrolysis probes that are labeled with the reporter dyes LightCycler® Red 610, LightCycler® Red 640, -LightCycler® Cyan 500, FAM or HEX (or any other dye that matches the emission and detection filters of the LightCycler® 480 Instrument). The LightCycler® 96 Instrument can detect hydrolysis probes that are labeled with the reporter dyes FAM, VIC, HEX, Yellow 555, LightCycler® Red 610, Texas Red and Cy5. These labeled hydrolysis probes can be used separately or in combination, which permits either single- or multicolor detection.

For multicolor hydrolysis probe assays it is recommended to use dark quencher dyes (i.e., dye molecules which efficiently quench the fluorescence of a FRET reporter dye without emitting fluorescence themselves). Roche recommends to use BHQ-2 (quenching range 550 -650 nm) for all hydrolysis probe reporter dyes listed above.
", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "The kit provides reagents, including an RNA master mix (with buffer, nucleotides, and enzyme), a Mn(OAc)2 stock solution, PCR grade water, and an enhancer solution. The LightCycler® 480 RNA Master Hydrolysis Probes can be used in conjunction with LightCycler® Uracil-DNA Glycosylase, heat-labile, for carry-over prevention during PCR. It provides convenience, high performance, reproducibility, and minimizes contamination risk. Only template RNA, PCR primers, hydrolysis probe, and Mn(OAc)2, need to be added.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "Easy-to-use Reaction Mix for One-Step RT-PCR using the LightCycler® 480 or LightCycler® 96 Real-Time PCR Systems.", "Country": "XG", "Code": "Product Characteristics", "Name": "Product Characteristics" }, { "Language": "en", "Value": "Hot Start

The hot start feature 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 reverse transcription (RT) and PCR in a single-tube reaction.
Aptamers are dedicated oligonucleotides that bind to 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 reaction setup and the subsequent 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. The recommended incubation temperature for RT with Tth DNA Polymerase (+61°C) can overcome secondary structures of RNA. This results in highly specific and efficient cDNA synthesis, which 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.


Hydrolysis Probe Assays

Hydrolysis probe assays use a single probe containing two labels: a fluorescent reporter dye and a quencher. When the probe is intact, the quencher is close to the reporter dye and suppresses the reporter fluorescence. When the probe is hybridized to the target sequence, the 5'-nuclease activity of the polymerase cleaves the hydrolysis probe, separating the reporter and quencher. With an increasing amount of target sequence during PCR, more probe is cleaved, and the fluorescence signal of the unquenched reporter dye increases.", "Country": "XG", "Code": "Background Information - Help Corner", "Name": "Background Information - Help Corner" }, { "Language": "en", "Value": "
VialCapLabelFunctionContent
1redLightCycler® 480 RNA Master Hydrolysis Probes, 2.7x conc.Contains Tth DNA Polymerase, reaction buffer and dNTP mix (with dUTP instead of dTTP).5 vials, 740 μl each
2colorless, grey cap labelActivator, 50 mMTo adjust Mn (OAc)2 concentration.1 ml
3colorlessWater, PCR gradeTo adjust the final reaction volume.5 vials, 1 ml each
4yellowEnhancer, 20x conc.To improve the amplification of difficult (e.g., GC-rich) targets.500 μl

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "The LightCycler® 480 RNA Master Hydrolysis Probes is designed for research studies. When combined with the LightCycler® 480 or LightCycler® 96 System, this kit uses a hot start RT-PCR protocol to provide very sensitive detection and quantification of defined RNA sequences (if suitable PCR primers and hydrolysis probe are supplied).
", "Country": "XG", "Code": "Intended Use", "Name": "Intended Use" } ] } } ] }

LightCycler® 480 RNA Master Hydrolysis Probes

Easy-to-use Reaction Mix for One-Step RT-PCR using the LightCycler® 480 or LightCycler® 96 Real-Time PCR Systems.

LightCycler<sup><sup>®</sup></sup> 480 RNA Master Hydrolysis Probes

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