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Standard Laboratory Equipment
  • Nuclease-free, aerosol-resistant pipette tips
  • Sterile reaction tubes for preparing master mixes and dilutions
For RT-PCR
  • LightCycler® Carousel-Based System*
  • LightCycler® Capillaries*
  • Standard benchtop microcentrifuge, containing a rotor for 2.0 ml reaction tubes
The LightCycler® Carousel-Based System includes Centrifuge Adapters that enable LightCycler® Capillaries to be centrifuged in a standard microcentrifuge rotor.
or
  • LC Carousel Centrifuge 2.0* for use with the LightCycler® 2.0 Sample Carousel (20 μl; optional)

  • Uracil-DNA Glycosylase, heat-labile* (optional)
For prevention of carryover contamination; see section Prevention of Carryover Contamination.

  • LightCycler® Color Compensation Set* (optional)
If you want to perform color compensation when using LightCycler® Red 640 and Cy5.5-labeled HybProbe pairs in dual-color experiments in the same capillary, see section Color Compensation.
", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "
Vial / BottleCapLabelFunction / DescriptionContent
1redLightCycler® RNA Master HybProbe, LC RNA Master HybProbe, 2.7x conc.Contains Tth DNA Polymerase, reaction buffer, and dNTP mix (with dUTP instead of dTTP).3 vials, 250 μl each
2colorlessLightCycler® RNA Master HybProbe, Mn(OAc)2 stock solution, 50 mMTo adjust the Mn(OAc)2 concentration.1 vial, 1 ml
3colorlessLightCycler® RNA Master HybProbe, Water, PCR GradeTo adjust the reaction volume.2 vials, 1 ml each
", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "For designing HybProbe probes, please refer to LightCycler® Technical Note 6/99 “Selection of Hybridization Probe Sequences for Use with the LightCycler”. In addition, LightCycler® Probe Design Software 2.0 can identify the best HybProbe probe and primer combinations providing precise estimations of the Tms of the primers and probes in LightCycler® Reagent Kits.



Recommended HybProbe Probes for the LightCycler® Carousel-Based System

The LightCycler® 1.5 Instrument has 3 channels: F1 [530], F2 [640] and F3 [705], for dual-color experiments using HybProbe probes labeled with LightCycler® Red 640 [640] and Cy5.5 [705].

The LightCycler® 2.0 Instrument has 6 channels: 530, 560, 610, 640, 670 and 705, for multi-color experiments using HybProbe probes labeled with LightCycler® Red 610, LightCycler® Red 640, Cy5 {670} and Cy5.5 {705}.



Difference between HybProbe probes and Hydrolysis/TaqMan® probes

HybProbe probes are not cleaved like hydrolysis probes, instead they are displaced. The ‘normal’ reaction of Taq DNA Polymerase during synthesis is to displace. The polymerase will cleave a probe only if it has certain characteristics and if the annealing/elongation temperature is +60°C, which is the temperature that the 5’-3’ exonuclease activity of the polymerase is optimal.



Performing a Melting Curve analysis with HybProbe probes can identify and characterize PCR products using their specific melting behavior for genotyping and mutation analysis.



The principle of Melting Curve analysis is:
  1. At the beginning of a melting curve analysis, the reaction temperature is low and the fluorescence signal is high.
  2. As the temperature steadily increases, the fluorescence will suddenly drop as the reaction reaches the melting temperature (Tm) of each DNA fragment.
  3. A graph of melting behavior [plot of the first negative derivative (-dF/dT) of the fluorescence vs. temperature] reveals that pure, homogeneous PCR products produce a single, sharply defined melting curve with a narrow peak. In contrast, primer-dimers melt at relatively low temperatures and have broader peaks.



Crossing Point (Cp)
  1. In an amplification reaction, the cycle at which the fluorescence of a sample rises above the background fluorescence is called the \"crossing point\" (Cp) of the sample.
  2. The Cp of a sample appears as a sharp upward curve on the display of the experiment's fluorescence chart.
  3. The Cp is the point (PCR cycle number) at which the amplified product is first visible.
  4. A sample's Cp depends on the initial concentration of DNA in the sample. A sample with a lower initial concentration of target DNA requires more amplification cycles to reach the Cp. A sample with a higher concentration requires fewer cycles to produce the Cp.

The software uses the calculated Cps of the standard samples to generate the standard curve of Cp versus sample concentration.", "Country": "XG", "Code": "Background Information - Help Corner", "Name": "Background Information - Help Corner" }, { "Language": "en", "Value": "
ProcedureAssay Time [min]
RT-PCR Setup15
Reverse Transcription20
LightCycler® Carousel-Based System PCR run (incl. Melting Curve)25
Total Assay Time60
", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "The LightCycler® RNA Master HybProbe is an easy-to-use hot start reaction mix, specifically adapted for one-step RT-PCR in LightCycler® Capillaries using the LightCycler® Carousel-Based System Instruments and HybProbe probes as the detection format.
Note: LightCycler® RNA Master HybProbe can be used in conjunction with heat-labile Uracil DNA-Glycosylase for carryover prevention during PCR.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "Easy-to-use Reaction Mix for One-Step RT-PCR, using HybProbe probes with the LightCycler® Carousel-Based System.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "Absolute fluorescence values in samples containing the same starting amount of template can vary. The most common reason is a difference in pipetting between samples. It is important to remember that absolute fluorescence readings for a sample are arbitrary and do not affect analysis of Cp (crossing point) determination or melting curve analysis in later data analysis.



During the seek process, the first capillary position is always found by the instrument, however, the capillaries thereafter must exceed a certain fluorescence background level (the \"seek threshold\") to be recognized by the instrument. With HybProbe probes, the main reason why capillaries are not found during the initial seek process is that the fluorescent dye has either been omitted or degraded. To prevent degradation of the HybProbe probes, keep them away from light!



Performing a 4-step Melting Curve can help when both probes have very different melting temperatures, to ensure that the probes bind correctly and that enough probe is bound for detection.
An example of a 4-step Melting Curve may be as follows:
+95°C for 0 sec
+59°C for 15 sec
+45°C for 15 sec
+95°C for 0 sec with a Ramp Rate of 0.1°C/sec and continuous acquisition.", "Country": "XG", "Code": "Troubleshooting - Help Corner", "Name": "Troubleshooting - Help Corner" }, { "Language": "en", "Value": "During cycling, Fluorescence readings for HybProbe probes should be taken at the end of annealing. In the LightCycler® Software, the Acquisition Mode should be ‘Single’ in the Annealing step.

For Melting Curve, the Acquisition Mode can be ‘Continuous’ or ‘Step’.

In the ‘Continuous’ mode, fluorescence data is acquired continuously as the temperature changes, with the rate of data acquisition dependent on the number of capillaries.

In the ‘Step’ mode, fluorescence data is acquired at each temperature transition. For example, if the Ramp Rate is 0.5°C/sec, fluorescence data is acquired every 0.5°C for every capillary.



Channel Settings

To correct for pipetting errors in assays using HybProbe probes, as the variation will occur in both channels, a division by the non-reporting signal from Channel 530 will compensate for potential difference between samples and standards.
  • The 530 option uses the values of every single data point in Channel 530 to correct the respective data point in the red fluorophore channel.
  • The Back 530 option refers to the signal from Channel 530 in cycles 2 to 6, thus this option is used for dual- or multi-color experiments. Due to the presence of several Fluorescein labeled probes in dual- and multi-color experiments, the corresponding signals from channel 530 can not be used to correct the signals of the reporter channels.

For amplification analysis of HybProbe probes, the channel setting should be divided by ‘530’ for mono-color experiments and by ‘Back 530’ for dual- or multi-color experiments.

For Melting Curve analysis of HybProbe probes, it is not necessary to divide by ‘530’ or ‘Back 530’.

For further information regarding Channel Settings, please refer to the LightCycler® Carousel-Based System LightCycler® Software 4.1 Addendum 1 - February 2010.", "Country": "XG", "Code": "Protocols - Help Corner", "Name": "Protocols - Help Corner" }, { "Language": "en", "Value": "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:
  1. 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.Fig. 1: Emission spectra of fluorescent dyes.
  2. 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.Fig. 2: Principle of the Residual Protein Trypsin ELISA.
  3. After annealing, an increase in temperature leads to elongation and displacement of the probes.Fig. 2: Principle of the Residual Protein Liberase ELISA.
  4. 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 Works
LightCycler® 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.
", "Country": "XG", "Code": "Principle", "Name": "Principle" } ] } } ] }

LightCycler® RNA Master HybProbe

Easy-to-use Reaction Mix for One-Step RT-PCR using the LightCycler® Carousel-Based System

LightCycler<sup><sup>®</sup></sup> RNA Master HybProbe

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