The LightCycler 480 System Short Guide Topic: Purpose: Assay Principle: Detection Format: Result: Relative Quantification (Mono-Color) Describes how to set up and perform mono-color Relative Quantification assays and how to analyze the data using basic or advanced methods, e.g., for gene expression studies. The expression level of a target sequence (relative to reference gene) in unknown samples is determined relative to a calibrator sample. Quantitative detection of nucleic acid sequences based on Universal ProbeLibrary (FAM-labeled, LNA-modified Hydrolysis Probe). * Automatic calculation of normalized ratios by using the Basic Analysis (ΔΔC T -Method). With Advanced Analysis the -Method (Efficiency Method) is provided, which can be used for customized settings and efficiency consideration. Results can be displayed as bar chart, sample view and result table. * Please note: This quick guide uses the Universal ProbeLibrary as an example. Other detection formats like SYBR Green I (together with LightCycler 480 SYBR Green I Master), or hybridization probes, and hydrolysis probes (both together with LightCycler 480 Probes Master) can be used for quantitative assays. Just select the appropriate run protocol of the template list provided in the LightCycler 480 Software database and follow the described procedure. Be aware that SYBR Green I assays require an additional proof of specificity, usually achieved by Melting Curve analysis. Requirements System LightCycler 480 System (96 or 384) LightCycler 480 Software 1.5 LightCycler 480 Multiwell Plate 96 or 384, white or clear Reagents Transcriptor First Strand cdna Synthesis Kit and LightCycler 480 Probes Master, or LightCycler 480 RNA Master Hydrolysis Probes Unknown samples (purified total RNA, mrna or cdna or genomic DNA) Sequence-specific forward and reverse primer sets for target and for reference gene(s) UPL Probes labeled with FAM dye to detect target and reference sequences For detailed information on Relative Quantification please refer to the LightCycler 480 Operator s Manual, section Relative Quantification Analysis.
Procedure Overview time Prepare reaction mix and set-up multiwell plate 15-60 Select protocol and edit samples 5-25 Perform PCR using LightCycler 480 Instrument 60-75 Analyze data Basic ( C T -Method) Advanced ( -Method) 5 Dummy Generate report 2 Relative Quantification (Mono-Color)
Preparing the Reaction Mix and Setting up the Multiwell Plate High quality cdna and primers are essential for good results in real-time PCR. Use the Transcriptor First Strand cdna Synthesis Kit for reverse transcription of RNA into cdna. For details refer to the instructions provided with the kit. Order highly purified primers only. Design assay specific primer/upl sets at http://www.universalprobelibrary.com for target and for reference gene real-time PCR. Prepare 10x conc. (2 µm each) solutions that contain either specific PCR primers for the target sequence or for the reference sequence. In two 1.5 ml reaction tubes on ice prepare two PCR mixes, one for the target amplification and one for the reference amplification. For one 10 µl reaction add the following componentes in the order listed below: PCR Mixes, separate for target and reference genes (without template nucleid acid) Reagent 1 Reaction Final Concentration LightCycler 480 Probes Master, 2x conc. 5 µl 1x Primer Mix, 10x conc. 1 µl 200 nm Selected Universal ProbeLibrary Probe, 10 µm 0.1 µl 100 nm Water, PCR-grade 1.4 µl Volume 7.5 µl 1. Pipet 7.5 µl PCR Mix into the respective wells of the LightCycler 480 Multiwell Plate. For each sample two wells are required: one for the target and one for the reference reaction. 2. Add 2.5 µl cdna template of all unknown samples. 3. Add 2.5 µl water for the No-Template-Control (NTC). Cover the multiwell plate with a sealing foil; use an adhesive seal applicator for correct sticking. Centrifuge the multiwell plate at 1,500 g for two minutes. Load the multiwell plate into the LightCycler 480 Instrument. Alternatively you can perform an one-step real-time PCR: a combination of reverse transcription and amplification in one run. Use LightCycler 480 RNA Master Hydrolysis Probes and follow the pack insert instruction for PCR setup and programming. Relative Quantification (Mono-Color) 3
Selecting the Protocol Change to the Overview screen by clicking. Click New Experiment from Template. Select Mono Color Hydrolysis Probe/UPL Probe for detection of FAM and set the reaction volume to 10 µl. Editing Samples Use the Subset Editor if necessary, to define subsets of samples subdividing the multiwell plate into several areas which can be analyzed separately. In all cases the full plate (all samples) will be measured. Use the Sample Editor to record information about the samples in the experiment. You can enter sample information manually before, during or after an experiment is completed. Step 1: Select the workflow Rel Quant. Step 2: Select the samples you want to edit. Step 3: Configure the properties. Here you have several possibilities: Edit sample by sample: Select one sample and and edit all associated properties Edit all samples with common properties: Select all samples with a common property, e.g., all samples with the property target unknown. Editing this property in the Step 3: Edit Rel Quant Properties fields changes all selected samples. Import all properties from a.txt file. For details please refer to the LightCycler 480 Operator s Manual, section Importing and Exporting Sample Information. 4 Relative Quantification (Mono-Color)
Properties Property Possible Values Description Sample Name Name of the sample, e.g., Hela 157/2 Please note: The sample name is used an identifier for the autopairing function from target to reference. Use the identical sample name for the same material, irrespective of wether the well is used as target or as reference. Combined Sample and Target Type Target Name Efficiency Target Unknown Target PosCalibrator Target Negative Target Standard Ref Unknown Ref PosCalibrator Ref Negative Ref Standard Unassigned Unknown Unassigned PosCal Unassigned Neg Unassigned Std Sample for measuring the target gene (gene of interest, GOI). You can normalize the results to the measured value of this sample. Negative control of your GOI. Standard sample with a known absolute or relative concentration. For using the Advanced Relative Quantification analysis method with an In-run standard curve, the Sample Editor needs to be expanded by the sample type Standard with the corresponding standard concentration values. Enter the concentration in the field Concentration. Sample for measuring the corresponding reference gene. You can normalize the results to the measured value of this sample. Negative control of your reference gene. Standard sample with a known absolute or relative concentration. The concentration is entered in the field Concentration. All samples with the type Unassigned are excluded from the analysis. For the target: Name of your GOI, e.g., Gene XY For the reference: Name of your Reference Gene, e.g., GAPDH If you do not want to create a standard curve and if the efficiency is known from a former experiment, enter here the appropriate value. Start Run Check if the multiwell plate is loaded into the LightCycler 480 Instrument. Click Start Run and name the experiment. Relative Quantification (Mono-Color) 5
Analysis The LightCycler 480 software provides two different analysis modes for Relative Quantification approaches: Basic and Advanced analysis: The quick and easy Basic Analysis method, ideally suited for assays with perfect and optimal performance, offers a fully automated way to generate the result with just one click. The Advanced Analysis method is based on standard curves. The final result considers the actual PCR efficiencies of all target and reference genes involved in the calculation and therefore guarantees a higher degree of precision. Basic Analysis Method (e.g., ΔΔC T -Method) Target and reference on the same plate Full plate analyzed Assay set up Advanced Analysis Method (e.g., E-Method) Target and reference on the same plate or on different plates Full plate and/or subsets analyzed Fixed pairing rule Pairing Flexible, editable pairing rules Assay Calibrator and/or Study Calibrator Calibrator Assay Calibrator and/or Study Calibrator - Standards (target/reference) In run or external standards E = 2, or editable efficiency values Efficiency Standard curves (linear, non-linear, fixed dynamic range) E = 2, or editable efficiency values Fit Points method Cp analysis Second Derivative Maximum or Fit Points method Click Analysis in the Module bar and select the analysis method in the Create New Analysis dialog box. For data analysis following the -Method we recommend to use the default settings. 6 Relative Quantification (Mono-Color)
Please note: In Basic mode the Manual Pairing tab is not available. If you want to check the subordinate Absolute Quantification analysis select the appropriate target or reference gene and click Show Abs Quant. The LightCycler 480 software automatically pairs the corresponding pairs (target to reference, sample ratio to calibrator ratio). The final calculation is displayed as result table or as bar chart and sample view. If samples are defined as standard in the Sample Editor, the option Standards (In Run) is enabled by default. The calculation is based on the efficiencies specified by these standard curves ( -Method). The corresponding standard curve is displayed under Target Name - Show Abs Quant. Click Save in the Global Action bar to save the analysis. Report Create a report by clicking the Report button. Generate an analysis report containing general and detailed experiment information and analysis results. You can customize the report to include several result items. Print the report, or save the report as pdf file. Relative Quantification (Mono-Color) 7
Ordering Information Roche Applied Science offers a large selection of reagents and systems for life science research. For a complete overview of related products, please visit our special interest sites for the LightCycler 480 Real-Time PCR System: http://www.lightcycler480.com Software LightCycler 480 Software, Version 1.5 1 software package 04 994 884 001 LightCycler 480 LIMS Interface Module 1 software package 05 066 310 001 LightCycler 480 Gene Scanning Software 1 software package 05 103 908 001 LightCycler 480 Multiple Plate Analysis Software 1 software package 05 075 122 001 Disposables LightCycler 480 Multiwell Plate 96, white 50 plates with 50 sealing foils 04 729 692 001 LightCycler 480 Multiwell Plate 384, white 50 plates with 50 sealing foils 04 729 749 001 LightCycler 480 Multiwell Plate 96, clear 50 plates with 50 sealing foils 05 102 413 001 LightCycler 480 Multiwell Plate 384, clear 50 plates with 50 sealing foils 05 102 430 001 PCR Reagents LightCycler 480 High Resolution Melting Master 5 100 µl (500 reactions, 20 µl each) 04 909 631 001 LightCycler 480 SYBR Green I Master LightCycler 480 Probes Master 1 kit (5 100 reactions, 20 µl each) 1 kit (10 500 reactions, 20 µl each) 1 kit (5 100 reactions, 20 µl each) 1 kit (10 500 reactions, 20 µl each) 1 kit (1 5000 reactions, 20 µl each) 04 707 516 001 04 887 352 001 04 707 494 001 04 887 301 001 04 902 343 001 LightCycler 480 Genotyping Master 1 kit (4 96 reactions, 20 µl each) 04 707 524 001 LightCycler 480 RNA Master Hydrolysis Probe 1 kit (5 100 reactions) 04 991 885 001 Transcriptor First Strand cdna Synthesis Kit Isolation of Nucleic Acids 1 kit (50 reactions, including 10 control reactions) 1 kit (100 reactions) 1 kit (200 reactions) 04 379 012 001 04 896 866 001 04 897 030 001 High Pure PCR Template Preparation Kit 100 purifications 11 796 828 001 For technical support please contact: www.roche-applied-science.com For life science research only. The product is covered in-part by US 5,871,908, co-exclusively licensed from Evotec OAI AG.Parts of the Software used for the LightCycler 480 System are licensed from Idaho Technology Inc., Salt Lake City, UT, USA. This product is covered by one or more of U.S. 6,197,520, 6,303,305, 6,387,621, 6,503,720, 6,730,501 and corresponding claims in their non-u.s. counterparts, owned by Roche Diagnostics GmbH and/or licensed from Idaho Technology, Inc. This LightCycler 480 Real-Time PCR System is licensed under U.S. Patent 6,814,934 and corresponding claims in its non-u.s. counterparts and under one or more of U.S. Patents Nos. 5,038,852, 5,656,493, 5,333,675, 5,475,610, 5,602,756, 6,703,236, 7,238,517 or corresponding claims in their non-u.s. counterparts, for use in life science research, in vitro diagnostics and other applied fields. No rights are conveyed expressly, by implication, or by estoppel under any patent claims or for any other application. LIGHTCYCLER, HYBPROBE, TAQMAN and HIGH PURE are trademarks of Roche. Other brands or product names are trademarks of their respective holders. Published by Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany 2009 Roche Diagnostics All rights reserved. 05465028001 0209