Roche LightCycler 480 System Reference guide

Brand: Roche

Model: LightCycler 480 System

Type: Reference guide

The LightCycler

480 System

Short Guide

Topic:

Relative Quantiﬁcation (Mono-Color)

Purpose: 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.

Assay Principle: The expression level of a target sequence (relative to reference gene) in unknown samples is determined

relative to a calibrator sample.

Detection Format: Quantitative detection of nucleic acid sequences based on Universal ProbeLibrary

(FAM-labeled, LNA-modiﬁed Hydrolysis Probe).

Result: Automatic calculation of normalized ratios by using the Basic Analysis (ΔΔC

-Method).

With Advanced Analysis the -Method (Efﬁciency Method) is provided, which can be used for cus-

tomized 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

15-60‘

5-25‘

60-75‘

5‘

Prepare reaction mix and set-up multiwell plate

Select protocol and edit samples

Generate report

Perform PCR using LightCycler

480 Instrument

Analyze data

Advanced ( -Method)Basic (∆∆C

-Method)

Dummy

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 ampliﬁca- ►

tion. 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.

3Relative Quantiﬁcation (Mono-Color)

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 ana-

lyzed 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, irrespec-

tive of wether the well is used as target or as reference.

Combined Sample and

Target Type

Target Unknown Sample for measuring the target gene (gene of interest, GOI).

Target PosCalibrator You can normalize the results to the measured value of this sample.

Target Negative Negative control of your GOI.

Target Standard 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.

Ref Unknown Sample for measuring the corresponding reference gene.

Ref PosCalibrator You can normalize the results to the measured value of this sample.

Ref Negative Negative control of your reference gene.

Ref Standard Standard sample with a known absolute or relative concentration. The concentration

is entered in the ﬁeld Concentration.

Unassigned Unknown

Unassigned PosCal

Unassigned Neg

Unassigned Std

All samples with the type Unassigned are excluded from the analysis.

Target Name For the target: Name of your GOI, e.g., Gene XY

For the reference: Name of your Reference Gene, e.g., GAPDH

Efﬁciency 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.

5Relative Quantiﬁcation (Mono-Color)

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

-Method) Advanced Analysis Method (e.g., E-Method)

Target and reference on the same plate ►

Full plate analyzed ►

Assay set up

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. ►

7Relative Quantiﬁcation (Mono-Color)

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 1 kit (5 × 100 reactions, 20 µl each)

1 kit (10 × 500 reactions, 20 µl each)

04 707 516 001

04 887 352 001

LightCycler

480 Probes Master 1 kit (5 × 100 reactions, 20 µl each)

1 kit (10 × 500 reactions, 20 µl each)

1 kit (1 × 5000 reactions, 20 µl each)

04 707 494 001

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 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

Isolation of Nucleic Acids

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

05465028001 0209

Roche LightCycler 480 System Reference guide

Roche LightCycler 480 System Reference guide

Roche LightCycler 480 System Reference guide

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