RHODE ISLAND GENOMICS and SEQUENCING CENTER
ROCHE LightCycler 480 Real-Time PCR Instrument - Standard Operating Procedure
Quantitative Real-Time PCR (qPCR) Using SYBR Green
Purpose:
The Roche LightCycler 480 Real-Time PCR System is a 96 well-plate based real-time PCR platform that is
used for highly accurate qualitative and quantitative detection of nucleic acids and genotyping. Fluorescent
signals measured during cycling are correlated with the amount of PCR product in the reaction, allowing the
calculation of input copy number of the target nucleic acid. The unique silver thermal-block cycler provides
rapid temperature ramping and excellent well-to-well temperature homogeneity, with minimal inter-well
temperature variability across the entire multiwell plate. The LightCycler 480 instrument's xenon lamp emits
light over a broad wavelength range (430–630 nm). The five excitation and six emission filters of the
instrument can be used in any combination, facilitating the use of a variety of fluorescent dyes and detection
formats. Uniform data capturing across the entire multiwell plate, together with superior signal acquisition
rates, allows melting curve analysis at high resolution. It also eliminates the need for passive reference dyes
(e.g., ROX) for well-to-well signal normalization.
The LightCycler 480 software enables fast, highly accurate data generation. Customizable views facilitate
intuitive, fast navigation, and the sample editor allows easy programming, data capturing and analysis. The
software package comprises automated analysis modules for melting curve or endpoint-based genotyping, as
well as absolute and relative quantification. Experiments can quickly be started from ready-to-use templates.
Users can apply basic or advanced analysis modes for both gene expression and genotyping studies. Various
quantitative real-time PCR (qPCR) analyses, such as absolute and relative quantification analysis methods, and
subtypes of these techniques are implemented in the LightCycler 480 software.
The following protocol describes the basic operation of the LightCycler 480 instrument to quantify specific
gene transcripts using the LightCycler 480 SYBR Green I Master kit. Specifically, the protocol includes: I.
Nomenclature for real-time PCR applications, II. Preparing and Starting a LightCycler 480 Instrument Run,
III. LightCycler 480 software, IV. Programming the LightCycler 480 SYBR Green I protocol, V. Preparing
and running the qPCR reactions. Please refer to the LightCycler 480 Operation Manual and the LightCycler
Real-Time PCR Systems Application Manual for detailed descriptions and additional information on multiple
protocols.
I. MIQE NOMENCLATURE GUIDELINES:
We recommend that users follow the standardized MIQE nomenclature guidelines1 for publication of
quantitative real-time PCR experiments:
 qPCR: quantitative real-time PCR
 RT-qPCR: reverse transcription - qPCR
 Reference Genes: this replaces the term housekeeping genes
 Hydrolysis Probes: this replaces the term TaqMan probes
 FRET probe (fluorescence resonance energy transfer probe): a generic mechanism in which
emission/quenching relies on the interaction between the electron-excitation states of 2
fluorescent dye molecules.
 Dual Hybridization Probes: LightCycler-type probes
 Quantification Cycle (Cq): this replaces the terms threshold cycle (Ct), crossing point (Cp), and
take-off point (TOP). [Note: The Roche manual and software use the crossing point (Cp) term
while the Stratagene Mx3005P manual and software use threshold cycle (Ct).]
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Bustin et al. 2009. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR
Experiments. Clin Chem 55(4) 611-622.
II. OVERVIEW: PREPARING and STARTING a LightCycler 480 RUN
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ON/OFF switch is located on the right side of the power box on the back of the instrument.
Two status LEDs are located on the front of the instrument
Orange (flashing)/Orange (flashing) = Instrument is initializing
Green/Orange = Instrument is turned on. Instrument is ready. No plate loaded.
Green/Orange (flashing) = Plate is loading.
Green/Green = Instrument is turned on. Instrument is ready. Plate is loaded.
Green (flashing)/ Green (flashing) = Instrument is running.
1. Turn on the instrument. Double-click on LightCycler 480 SW1.5 icon.
2. In the LC480 Login dialog box enter:
User name = To be assigned by the RIGSC manager (users will also be assigned to a
Group associated with their research lab)
Password = Roche480 (change password as necessary)
Log on to = Research database
3. Program the experimental protocol and define the sample numbers, names, etc.
4. Prepare a master mix that contains all of the reaction reagents except the DNA template into a 1.5 ml or 0.5
ml reaction tube. Use dark reaction tubes to avoid bleaching of fluorescent dyes by ambient light.
5. Pipette the reagent mix into the wells of the LightCycler® 480 96-well plate. Robotic or parallel pipetting is
recommended when processing a large number of samples. For ease of analysis, standards should be loaded
sequentially in columns.
6. Add the DNA template to each well
7. Seal the plate properly with a LightCycler® 480 sealing foil by pressing it firmly to the plate surface using
your hand or a sealing foil applicator.
8. Place the multiwell plate in a standard swing-bucket centrifuge, containing a rotor for multiwell plates with
suitable adaptors. Balance with another multiwell plate. Centrifuge the plate at 1,500 × g for two minutes.
Check the wells for bubbles, and repeat if necessary.
9. To load the prepared multiwell plate into the instrument, press the push button on the front of the instrument
(located next to the instrument status LEDs)
 The multiwell plate loader extends out of the right side of the instrument
10. Place the multiwell plate into the loading frame of the loader with the flat edge (A-H) pointing towards the
instrument. The short plate edge with beveled corner points away from the instrument.
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11. Press the plate loading push button again to retract the loader with the inserted multiwell plate into the
instrument. Both LEDs should be green. You are now ready to start the run.
12. When the run is finished, open the plate loader to remove the plate then close the plate loader. Caution:
plate may be hot! Exit the LightCycler 480 software (through the red door!). TURN OFF the
instrument.
III. LightCycler® 480 SOFTWARE
1. Double-click the LightCycler® 480 icon on the desktop.
2. In the “Login” dialog box, type your user name and password and log on to the Research Database. Click
 to proceed.
 Display: “Overview” window containing the “Status” bar at the top, the “Experiment Creation”
and “Tasks” on the right, and the “Global Action” bar at the extreme right.
 Status bar: Information about the currently active object. Switch between open objects by
selecting them in the window list
 Experiment creation: Click one of the buttons to create a new experiment and launch the
Run module. Choose the plate type (clear or white).
 Tasks: Click “Open Existing Object” to open an object which already exists in the
database.
 Global Action bar: Availability of options depends on the active window currently opened.
 Message area: Located on bottom of screen, displays status messages, errors, and warnings.
 Editor Frame: The frame is the central area where the modules are displayed. It may
contain several sections that can be resized individually. You can resize a section by
dragging the splitter bar on the border between two sections to hide or show the section.
The arrows on the splitter bar indicate which area of the Editor frame will be affected.
Clicking the splitter bar will hide the corresponding area.
 Navigator: The Navigator window provides access to items stored in the LightCycler® 480
database. Items include experiments, instrument, macros, etc. The Navigator allows you to
open experiments and related items (such as preferences, macros, special data) as discrete
objects. All items in the Navigator are organized in folders in a tree-like structure (similar to
Windows Explorer) and are sorted alphabetically within their folders. You can expand and
contract folder views and highlight the object you want to select. In addition, you can use
the Query tab to search for specific LightCycler® 480 Software objects in the database by
entering search parameters. Note: The Navigator is similar, but not identical to the
folder/file tree in Windows Explorer. The Navigator displays data that are stored in a
database (Exor4) not in the Windows file system. These data files are NOT visible in the
Windows file system. Therefore, users may ONLY manage their data files through the
LC480 software.
3. Create “New Experiment” or “New Experiment from Template” in the Overview window.
Note: a general template has been created for SYBR Green I Master.
4. In the “Setup” area of the “Run Protocol” tab, specify the following setup parameters:
 Detection format: SYBR Green I
 Block Size: 96 (default setting)
 Plate ID: optional
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Reaction volume: 20 l (instrument range 10 – 100 l)
Color Comp ID: optional
Lot No.: optional
Test ID: optional
5. In the “Programs and Temperature Targets” section modify and add setup parameters, experiment programs,
and temperature targets as needed. Refer to specific protocols for exact program parameters. Note: The
program overview area on the bottom of the screen will display green dots along the temperature cycling
graph indicating that the instrument is ready.
6. In the “Module” bar, click “Subset Editor” to define sample subsets.
Note: You are not required to define sample and subset information before the run. The information can be
defined during or after a run.
 To enter information to calculate a standard curve, click “Sample Editor” in the “Module” bar and
select the workflow “Abs Quant”. Select “Standard” as the sample type for each standard and
specify the standard concentration (e.g. 1.00E2 = 102 copies).
7. Prepare the plate and load it in the instrument as described above.
8. Click “Start Run”. This option is only available if a plate has been loaded.
 The “Save Experiment” dialog is displayed. Enter a name for the experiment.
 Note: If you are using a clear plate, a warning message indicates that the plate detection sensor
is switched off. This is the correct setting for SYBR Green quantification.
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A status bar on the “Data” tab indicates the progress of the running experiment.
 As the experiment progresses, the “Messages” area displays messages indicating information,
warnings, and errors encountered during the run.
 Sample data is displayed in the charts on the “Data” tab.
9. To view data for specific samples, select one or more samples in the “Sample Editor” or “Sample Table”.
Chart settings can be modified during the experiment if needed.
10. To adjust or stop the program during the run:
 Click “End Program” to stop the current program and skip to the next program in the experiment
protocol. Analysis can be performed on the run.
 Click “+10” cycles to add 10 cycles to the current program.
 Click “Abort Run” to stop the run. The “Abort Run” button replaces the start button during the run.
Performing this task results in incomplete data, and no analysis can be performed.
11. When the experiment is finished, a status message displays “Run Complete”
12. To analyze your run:
 In the “Module” bar, click “Sample Editor”. Enter sample information if you have not already done so.
 Click “Analysis” on the “Module” bar to open the “Analysis Overview” window.
 The “Analysis Overview” window displays the “Create New Analysis” list and “Open Existing
Analysis” list (if an analysis was created before).
 For SYBR I quantification choose :
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Absolute quantification: allows you to quantify a single target sequence and express the final
result as an absolute value. Also displays the standard curve.
Note: You must click “calculate” to calculate the standard curve. You also must recalculate if
you change any information.
Tm Calling: gives the melting curve analysis.
13. To backup your data after completing the analysis, go to the Navigator and Export your experiment to a
safe place (a folder on the D-drive or your personal flash drive).
14. When the run is finished, open the plate loader to remove the plate then close the plate loader. Caution
plate may be hot! Exit the LightCycler 480 software (through the red door!). TURN OFF the
instrument.
IV. PROGRAMMING the LightCycler 480 SOFTWARE FOR SYBR GREEN I QUANTIFICATION
The LightCycler® 480 Instrument should be programmed prior to preparation of master mixes!
The LightCycler® 480 SYBR Green I Master template contains the following programs:
• Pre-Incubation for activation of FastStart Taq DNA polymerase and denaturation of the DNA
• Amplification of the target DNA
• Melting Curve for PCR product identification
• Cooling the multiwell plate
Note: The plate cooling temperature must never be set lower than 40°C!
The following table shows the PCR parameters that must be programmed for a LightCycler® 480 System PCR
run with the LightCycler® 480 SYBR Green I Master using a LightCycler® 480 Multiwell Plate 96.
1) 45 cycles are suitable for most assays. If the assay is optimized and has steep amplification
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curves and early crossing points (even when target concentrations are low), 40 cycles
should be sufficient. Reducing the number of cycles will reduce the time required for the assay!
2) For initial experiments, set the target temperature (i.e., the primer annealing temperature)
5°C below the calculated primer Tm.
3) If high polymerase activity is required in early cycles, you might improve results by extending
the pre-incubation to 10 min. Especially, apply such an extended pre-incubation time
when working with unpurified cDNA samples as template. Do not use more than 2 l
unpurified cDNA sample.
4) For greater precision in target quantification experiments, it can be advantageous (in some
cases) to choose longer annealing and extension times for the amplification cycles.
5) Calculate the hold time for the PCR elongation step by dividing the amplicon length over 25
(e.g., a 500 bp amplicon requires 20 s elongation time).
6) The Tm calculated by Tm Calling Analysis might differ approx. 0.5°C when using either the
lowest (5) or highest (10) possible value of Acquisitions/°C.
7) For users of LightCycler® 480 Software 1.1: (From LightCycler® 480 Software 1.2 on, the
Cooling Ramp Rate can be set to maximum.)
• For the 96-multiwell plate: For target temperatures of 50°C and above, set the Ramp Rate
for Cooling to 2.2°C/s. For target temperatures below 50°C, set the Ramp Rate to 1.5°C/s!
8) The Tm calculated by Tm Calling Analysis might differ by up to 2°C when using either the
lowest (10 l) or highest (100 l) possible reaction volume.
V. LightCycler 480 SYBR GREEN I MASTER MIX PROTOCOL
Please refer to the manual included with the kit for more detailed information. This kit is ideally suited for hotstart PCR applications. In combination with the LightCycler® 480 System and suitable PCR primers, this kit
allows very sensitive detection and quantification of defined DNA sequences. The kit can also be used to
perform two-step RT-qPCR. The kit can be used for the amplification and detection of any DNA or cDNA
target. However, you would need to adapt each amplification protocol to the reaction conditions of the
LightCycler® 480 Instrument and design specific PCR primers for each target. See the LightCycler® 480
Operator’s Manual for general recommendations. The amplicon size should not exceed 750 bp in length. For
optimal results, select a product length of 500 bp or less.
Generation of PCR products can be detected by measurement of the SYBR Green I fluorescence signal. SYBR
Green I intercalates into the dsDNA helix. In solution, the unbound dye exhibits very little fluorescence;
however, fluorescence (measured at 530 nm) is greatly enhanced (100-fold) upon binding to DNA due to
conformational changes. Therefore, during PCR, the increase in SYBR Green I fluorescence is directly
proportional to the amount of dsDNA generated.
Sample Material
 Use any template (e.g. genomic DNA, plasmid DNA, cDNA)
 Use up to 50 – 100 ng complex genomic DNA or up to 108 copies plasmid DNA for reaction volume of
20 l. For larger volumes, the amount of template can be increased equivalently.
 Using a too high amount of template DNA might reduce the maximum fluorescence signal by outcompeting the SYBR Green I dye.
 If you are using a non-purified cDNA sample from reverse transcription, especially if it contains high
background concentrations of RNA and oligonucleotides, you can improve your results by using a
maximum of 2 l of that sample and applying a 10 min pre-incubation at 95°C. This will result in lower
quantification cycle (Cq) values with a decreased standard deviation.
Standard Curve
For Absolute Quantification analyses used with SYBR Green I, serial dilutions of an external standard with
predefined known concentration are used to create a standard curve. The standard dilutions are amplified in
separated wells but within the same run. The Cq values of standards and unknown samples are then used to
determine the concentration of target DNA.
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A typical standard curve should contain at least five samples, which are prepared by serial dilution.
The concentrations chosen for the standard curve should fall between the expected concentration ranges
of the target.
The slope of the standard curve describes the kinetics of the PCR amplification. It is also referred to as
the “Efficiency” of the amplification reaction. A perfect amplification reaction would produce a
standard curve with an efficiency of “2”, because the amount of target nucleic acid would double with
each amplification cycle. The efficiency is automatically calculated and displayed on the analysis
window.
The “Error” value (mean squared error of the single data points fit to the regression line), given on the
left side of the standard curve, is a measure of the accuracy of the quantification result based on the
standard curve. An acceptable value should be <0.2.
By determining where an unknown sample’s quantification cycle (Cq) falls on the standard curve, the
software can determine the initial concentration of target DNA in the sample.
Negative Control
Always run negative controls with your samples. To prepare negative controls:
 Replace the template DNA with PCR-grade water (vial 2; this will reveal whether a contamination
problem exists)
 In a 2-step RT-qPCR setup, omit addition of reverse transcriptase to the cDNA synthesis reaction (this
will indicate whether DNA in RNA samples causes false-positive results)
Primers
Use PCR primers at a final concentration of 0.2 to 1 M. The recommended starting concentration is 0.5 M
each.
 The optimal primer concentration is the lowest concentration that results in the lowest Cq and an
adequate fluorescence for a given target concentration.
Experimental Protocol
Follow the procedure below to prepare one 20 l standard reaction.
 Program the LightCycler® 480 Instrument before preparing the reaction mixes! See section above to
learn how to program the LightCycler® 480 Instrument.
 The LightCycler® 480 uses specially designed clear or white 96-well plates. Use the clear plates only
with SYBR Green I, hydrolysis probes, or if specifically recommended.
 Do not touch the surface of the LightCycler® 480 Multiwell Plate and Multiwell Sealing Foil when
handling them!
 Always wear gloves during handling!
 Keep the Master mix away from light!
1. Thaw one vial of “LightCycler® 480 SYBR Green I Master” (vial 1, green cap) and Water, PCR-grade.
2. Prepare a 10× conc. solution of the PCR primers.
3. In a 1.5 ml reaction tube on ice, prepare the PCR Mix for one 20 l reaction by adding the following
components in the order mentioned below:
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To prepare the PCR Mix for more than one reaction, multiply the amount in the “Volume” column
above by z, where z = the number of reactions to be run + sufficient additional reactions.
If you use reaction volumes different from 20 l, be sure to adapt the right volume in the running
protocol. As a starting condition, we recommend to use the same hold times as for the 20 l volume.
Mix carefully by pipetting up and down. Do not vortex!
4. Pipette 15 l PCR mix into each well of the LightCycler® 480 CLEAR Multiwell Plate.
5. Add 5 l of the DNA template.
6. Seal the Multiwell Plate with LightCycler® 480 Multiwell Sealing Foil.
 Sealing the plate is crucial to eliminate evaporation at high temperatures.
7. Place the Multiwell Plate in the centrifuge and balance it with a suitable counterweight (e.g., another
Multiwell Plate).
 Centrifuge at 1500 × g for 2 min (3000 rpm in a standard swing-bucket centrifuge containing a rotor for
multiwell plates with suitable adaptors).
8. Load the Multiwell Plate into the LightCycler® 480 Instrument.
9. Start the PCR program described above.
Two-Step RT-qPCR
LightCycler® 480 SYBR Green I Master can also be used to perform two-step RT-qPCR. In two-step RTqPCR, the reverse transcription of RNA into cDNA is separated from the other reaction steps and is performed
outside the LightCycler® 480 System. Subsequent amplification and online monitoring is performed according
to the standard LightCycler® 480 System procedure, using the cDNA as the starting sample material.
Transcriptor First Strand cDNA Synthesis Kit* is recommended for reverse transcription of RNA into cDNA.
Synthesis of cDNA is performed according to the detailed instructions provided with the kit. For initial
experiments, we recommend running undiluted, 1:10 diluted, and 1:100 diluted cDNA template in parallel to
determine the optimum template amount. If you use undiluted cDNA as template, we recommend extending the
pre-incubation time to 10 minutes.
Melting Curve Analysis
To prove that only your desired PCR product has been amplified, you may perform a melting curve analysis
after PCR. In melting curve analysis the reaction mixture is slowly heated to 97°C, which causes melting of
double-stranded DNA and a corresponding decrease of SYBR Green I fluorescence. The instrument
continuously monitors this fluorescence decrease and displays it as melting peaks. Each melting peak represents
the characteristic melting temperature (Tm) of a particular DNA product (where the DNA is 50% double
stranded and 50% single-stranded). The most important factors that determine the Tm of dsDNA are the length
and the GC-content of that fragment. If PCR generated only one amplicon, melting curve analysis will show
only one melting peak. If primer-dimers or other non-specific products are present, they will be shown as
additional melting peaks. Checking the Tm of a PCR product can thus be compared with analyzing a PCR
product by length in gel electrophoresis.
[Created by Maureen Varina, July 2009]
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