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For Professional Use Only
GUIDELINES
toAmpliSens HPV6/11-FRTPCR kit
for qualitative detection and differentiation of types 6 and 11
ofhuman papillomavirus (HPV)DNA in clinical material by the
polymerase chain reaction (PCR) with real-time hybridization-
fluorescence detection
Ecoli s.r.o., Studenohorska12841 03 Bratislava 47
Slovak RepublicTel.: +421 2 6478 9336Fax: +421 2 6478 9040
Federal Budget Institute ofScience Central ResearchInstitute for Epidemiology
3A Novogireevskaya StreetMoscow 111123 Russia
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TABLE OF CONTENTSINTENDED USE .................................................................................................................. 3AMPLIFICATION AND DATA ANALYSIS WITH Rotor-Gene 3000/6000 (CorbettResearch, Australia) INSTRUMENTS ................................................................................. 3AMPLIFICATION AND DATA ANALYSIS WITH iCycler iQ5 (Bio-Rad, USA)INSTRUMENTS ................................................................................................................. 11
AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA)INSTRUMENT ................................................................................................................... 16
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INTENDED USE
Guidelines describe the procedure of the use of AmpliSens
HPV 6/11-FRTPCR kitfor
qualitative detection and differentiation of types 6 and 11 ofhuman papillomavirus (HPV)
DNA by means of real-time hybridization-fluorescence detection with the following real-time PCR instruments:
Rotor-Gene 3000, Rotor-Gene 6000 (Corbett Research, Australia),
iCycler iQ5 (Bio-Rad, USA),
Mx3000P (Stratagene, USA).
AMPLIFICATION AND DATA ANALYSIS WITH Rotor-Gene 3000/6000 (Corbett
Research, Australia) INSTRUMENTS
Carry out sample pretreatment and reaction mixture preparation stages according to the
instruction manual. Transparent no domed 0.2-mlPCR tubes (detection through the
bottom of a tube) or 0.1-ml tubes are recommended for use.
Hereinafter, all terms corresponding to different instruments and software are
indicated in the following order: for Rotor-Gene 3000/for Rotor-Gene 6000.
Whenworking with Rotor-Gene 3000 instrument use the program Rotor-Gene version 6.
When working with Rotor-Gene 6000 instrument use the program Rotor-Gene 6000
versions 1.7 (build 67) or higher.
Programming the instrument
1. Turn the instrument on and start Rotor-Gene program.
2. Place the tubes in the rotor so that the first tube is in No. 1 well, insert the rotor in the
reaction module, and secure the cover (the rotor cells are numbered and these
numbers are used for sample order programming).
Balance the rotor if it is not loaded entirely. Fill unused wells with empty tubes. Do
not use tubes from previous runs.No.1 well should be loaded with a test tube from the current experiment (notempty).
3. Program the instrument to run amplification and detection:
In the opened window select Advancedmode. Activate the Newbutton.
Select 36-Well Roto r (or 72-Well Roto r) and No Domed Tubes/Lock ing r ing
attached. Click Next.
In the opened window define the operator and set the reaction volume: React ion
vo lumeis 25 l. Tick off the 15loi l layer vo lum eoption.
set amplification program.
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Table 1
DNAHPVtypes 6 and 11 amplification program for
Rotor-Gene 3000/6000 (Corbett Research, Australia)
Stage emperature, TimeFluorescence
detection
Cycle
repeatsHold 95 15 min 1
Cycling
95 15 s
4560 35 s
FAM/Green,JOE/Yellow,ROX/Orange
This program can be applied for both AmpliSensHPV6/11-FRTandAmpliSensHPV16/18-FRTPCR kits.
Following amplification programs can be used as well:AmpliSens-1 RG (see
Table 2) and amplification program for HPVHCR DNA types 16, 18, 31,33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 3) (this program is applied forAmpliSensHPVHCR screen-titre-FRT PCR kit). AmpliSens-1 RGamplification program is a universal program and it can be used for runningdifferent tests within one run (for example tests for detection of STIs). Fordetection of DNA ofHCRHPV and HPVDNA types 6, 11 within one runAmpliSens FRT HR HPVScreenRG4x Program can be applied.
Using amplification program for HPVHCR DNA types 16, 18, 31, 33, 35,39, 45, 51, 52, 56, 58, 59 (from AmpliSensHPVHCR screen-titre-FRT PCRkit) does not affect analytical performances of this PCR kit.
Table 2
AmpliSens-1 RG amplification program
Step Temperature, TimeFluorescence
detection
Cycle
repeats
Hold 95 15 min 1
Cycling 1
95 5 s
560 20 s
72 15 s
Cycling 2
95 5 s
4060 20 s
FAM/Green,
JOE/Yellow,ROX/Orange,
Cy5/Red
72 15 s
Cy5/Red detection channel should be enabled for running multiplex tests (ifrequired).
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Table 3
Amplification program for HPVHCR DNA types16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
Step Temperature, TimeFluorescence
detectionCycle repeats
Hold 95 15 min 1Hold 2 65 2 min 1
Cycling
95 20 s
564
Touchdown:1 deg. per cycle
25 s
65 55 s
Cycling 2
95 15 s
40
60 25 s
65 25 s
FAM/Green,JOE/Yellow,
ROX/Orange,Cy5/Red
The file AmpliSens FRT HR HPVScreen RG4x Program.proenclosed toAmpliSensHPVHCR screen-titre-FRT PCR kit can be used for programming.
Click the Calibrate/Gain Opt imis at ionbutton in the New Run Wizardwindow.
a) for detection in FAM/Green, JOE/Yellow, ROX/Orange, and Cy5/Redchannels
select Calibrate Acq uir ing /Opt imis e Acq uir ing;
b) click Perform Calibrat ion Before 1st Acquis i t ion/Perform Opt imisat ion
Before 1stAcquis i t ion.
c) click the Edit button in the Auto ga in ca l ib rat ion ch annel set tings window
and set Min Reading as4andMax Reading as8for all channels.
4. Select the Start runbutton for amplification to run. Name the experiment and save it to
the disk (results of the run will be automatically saved in this file).
5. Enter data in the table of samples (opens by default when the run begins). Define
names/numbers of clinical samples in the Namecolumn. Define the Negative Control
of amplification as NCA andthe Positive Control of amplification as C+. Enter the
Unknown type for all clinical and control samples. Indicate the None type for empty
wells.
The sample will not be analyzed if its type is defined as None.
Due to the fact that the PCR kit applies a multiplex control including several DNA matrixes
(HPVtype 6, HPVtype 11 and human DNA) several pages should be created in protocol.
To do this, indicate HPV 6(FAM/Green channel) in the Pagearea of the Name field. Click
New. A new page for information about samples will appear. It contains the same names
of samples as in previous page. Indicate HPV 11(JOE/Yellow channel) in thePagearea
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TROUBLESHOOTING
Review this section before running the experiment
Problem Cause How to identify Ways to eliminateNegative samples areanalyzed as positive
samples by Rotor-Gene program
Incorrectmathematical
processing ofnegative samples inthe presence of dropof fluorescence atthe initial cycles(Fig. 2a)
A normal positivesample has a S-
shaped curve offluorescenceaccumulation (Fig. 1,3-5). The negativesamples processedincorrectly appear asquite straight bottom-up lines (Fig. 7)
Use the Ignore Firstoption: select
5 cycles. If it does nothelp, increase thisvalue by 1-5
The threshold linecrosses descendingfluorescence curvesat the initial cycles
(Fig. 6)
The red threshold linecrosses (or touches)fluorescence curvesat the left (initial
cycles) on the chart ofprocessedfluorescence curves(Fig. 6)
Use the Eliminatecycles beforeoption: select 5(crossing the
threshold withfluorescence curve isignored for the first 5cycles)
Drop of sensitivity dueto contamination ofinstrument lenses
Lensescontamination leadsto reduction ofeffectiveness offluorescenceexcitation anddetection. Primarilythis has an impact
on the samplescontaining smallquantity of specificDNA which showlow fluorescencegrowth
Low backgroundsignal in all fourdetection channels(
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increase can appearafter cleansing theinstrument lensesfrom severcontamination
Desensitization due to
decrease ofpolymerase (TaqF)activity
Incorrect storage or
contamination causeenzymedeterioration
It appears as a fail of
the positive control orif a boundary Ct valueof the positive controlis greater than thethreshold of weaksamples
Use the reagent
stored under requiredcondition (see partStability and Storagein the InstructionManual) or use a newreagent
Note! A report on all parameters specified as well as a report on auto-calibration isavailable if the Sett ings button is activated. In particular, the Messages tab, theAutoca l ib ra tion Log Messages option.
Problem Feature Ways to eliminateContamination of a specificDNA
Detection of a signal of thenegative control in anychannel
Repeat the experiment. Takemeasures to detect andeliminate the source ofcontamination
The volume of a DNA sampleadded to a reaction tube isless than required/DNAsamples is not added
Background signal of asample is much stronger thanthe others (see raw curves,Fig. 2b). The sample isnegative
Repeat PCR for this sample
The volume of the reactionmixture added to a reaction
tube is less thanrequired/reaction mixture isnot added/the volume of aDNA sample added to areaction tube is greater thanrequired
Background signal of asample is much lower than the
others (see raw curves, Fig.2b).
If the sample is negative,repeat the test
The autocalibration parameterf rom 4Fl to 8Flis not set or itis a failure of the first tube inthe rotor (the No.1 well isempty; a volume of DNAsample or reaction mixture
added to the tube is incorrect)
Most of the fluorescencebackground signals are lessthan 1 or greater than 20
Set the parameter in the nextrun. In case of off-scalereading or if the signals aretoo weak (no positive signalsafter processing; abackground is less than 0.5)
the experiment should berepeated beginning with PCR
Polymerase (TaqF) was notadded to a reaction mixture
Positive signals are absent forall samples including apositive control
Repeat the experimentbeginning with PCR. Ensureappropriate preparation ofreaction mixtures
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Unprocessed data (raw channel)
Fig. 1. Raw curves are normalFig. 2. Raw curves have a bend (drop offluorescence at the initial cycles)
Fig. 2b. Level of background signal points on thepossible error:DNA sample was not added to the tube 1;double volume of DNA sample was added to the
tube 2
Processed data (Quantitation analysis)
) processed curves are normal (S-shaped, threshold line crosses curves at the area of
growth of fluorescence)
Fig. 3. FAM channelFig. 4. JOE channel
2
1
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Fig. 5. ROX channelinternal control(-globin gene)
b) curves are processed incorrectly
Fig. 6. Threshold line crosses fluorescencecurves twice
Fig. 7. Incorrect processing of the curves withbend (from Fig. 2)
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AMPLIFICATION AND DATA ANALYSIS WITH iCycler iQ5 (Bio-Rad, USA)
INSTRUMENTS
Carry out sample pretreatment and reaction mixture preparation stages according to the
instruction manual. Transparent domed 0.2-mlPCR tubes (detection through the cap of
the tube) are recommended for use.
1. Switch on the instrument and the power supply of the optical module.
The lamp should be warmed up for at least 15 min before the experiment starts.
2. Open the iQ5 program.
Table 4
DNAHPVtypes 6 and 11 amplification programfor iCycler iQ, iQ5 (Bio-Rad, USA)
Step Temperature, TimeFluorescence
detection
Cycle
repeats
1 95 15 min 1
295 20 s
4560 1 min FAM, HEX, ROX
This program can be applied for both AmpliSens
HPV6/11-FRTandAmpliSensHPV16/18-FRTPCR kits.
Following amplification programs can be used as well:AmpliSens-1 iQ (seeTable 5) and amplification program for HPVHCR DNA types 16, 18, 31,33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 6) (this program is applied forAmpliSensHPVHCR screen-titre-FRT PCR kit). AmpliSens-1 iQ amplificationprogram is universal program and it can be used for running different testswithin one run (for example tests for detection of STIs). For detection of DNAof HCR HPV and HPV DNA types 6, 11 within one run AmpliSens FRT HRHPV ScreenRG4x Program can be applied.
Using amplification program for HPVHCR DNA types 16, 18, 31, 33, 35,
39, 45, 51, 52, 56, 58, 59 (from AmpliSens
HPVHCR screen-titre-FRT PCRkit) does not affect analytical performances of this PCR kit
Table 5
AmpliSens-1 iQ amplification program
Step Temperature, TimeFluorescence
detection
Cycle
repeats
1 95 15 min 1
2
95 5 s
560 20 s
72 15 s
3
95 5 s
4060 30 s FAM, HEX, ROX, Cy5
72 15 s
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Cy5/Red detection channel should be enabled for running multiplex tests (ifrequired).
Table 6
Amplification program for HPVHCR DNA types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
Step Temperature, TimeFluorescence
detection
Cycle
repeats
Cycle 1 95 15 min 1
Cycle 3
95 15 s
665
Touchdown:1 deg. per cycle
55 s
65 25 s
Cycle 4
95 15 s
4160 55 s Real-time
65 25 s
The AmpliSens FRT HR HPV Screen iQ5.tmo file enclosed to AmpliSensHPVHCR screen-titre-FRTPCR kitcan be used for programming.
Create a new plate setup in the Edit Plate Setup tab. Indicate samples as Unknown.
Proceed to the Select fluoro forestab and set fluorescence detection for all tubes in the
FAM, HEX and ROX channels. Go back to the Samp les: Whole plate loadingand click
the Per Dye Layerbutton.
Click Run. Select Well factor (both well factor by experiment tubes and fixed well factor
are acceptable for use but the latter is recommended). Run the experiment.
Data analysis
1. Proceed to thePCR Quant i f icat iontab in theData Analysismode.
2. Review data in one channel at a time.
3. Ensure that the threshold line was set correctly for each channel. If it is required,
activate User Def ined, 2 through 10 cycles parameter. Normally threshold line
should cross s-shaped curves of signal accumulation of positive samples and controls
and not cross the base line. Otherwise, raise the threshold line up to the level where it
crosses positive samples only.
4. Activate the Resultsbutton (below the fluorofore buttons).
5. Proceed to the PCR Standard Curv etab, review parameters of the reaction, copy the
results.
6. Click on the results grid using the mouse right button. Select Export to Excelfrom the
drop-down menu. Agree to save the file. If Microsoft Excel program is loaded on the
computer it will open automatically (Microsoft Excel program is required for further
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data analysis). Results in the FAM, HEX, ROX, and then Cy5 channels follow
consistently.
TROUBLESHOOTING
Problem Cause How to identify Ways to eliminate
Drop of sensitivity dueto deterioration ofprobes
Incorrect storage orutilizing kitcomponents (hightemperature,multiple openingreagent tubes, foulworking conditions)can cause destroy ofoligonucleotides
Probe destruction canbe find out as a resultof comparison ofexperimental dataobtained at the firstuse of the reagentsand after a period oftime or in comparisonwith the reagents ofthe same lot storedproperly. It isappeared as an
increase ofbackgroundfluorescence(fluorescence isassessed in theBackground
subtractedmode atthe beginning of theexperiment) twice asmuch in differentexperiments (in casethe same instrument
is used). Note!Effectof f luorescenceincrease can appearin case the lamp waschanged, opticalsystem was cleaned,or calibration wasdone)
Use mixtures storedunder statedconditions untillabeled expire date(see part Stabilityand Storage in theInstruction Manual)
Drop of sensitivity dueto decrease of activityof polymerase (TaqF)
Incorrect storage orcontamination causeenzyme
deterioration
It appears as a fail ofthe positive control orif a boundary Ct value
of the positive controlis much greater thanthe normal one
Use the reagentstored under requiredcondition (see part
Stability and Storagein the InstructionManual) or use a newreagent
Problem Feature Ways to eliminate
Contamination of a specificDNA
Detection of a signal for anegative control in anychannel
Repeat the experiment. Takemeasures to detect andeliminate the source ofcontamination
The volume of a DNA sampleadded to a reaction tube is
less than required/DNAsamples is not added
Background signal of thesample is much stronger than
the others (see raw curvesBackground subtractedmode). The sample isnegative
Repeat PCR for this sample
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The volume of a reactionmixture added to a reactiontube is less thanrequired/reaction mixture isnot addedorthe volume of aDNA sample added to a
reaction tube is greater thanrequired
Background signal of thesample is much lower than theothers (see raw curves-Background subtractedmode)
If the sample is negative,repeat the test
Incorrect adjustment of thethreshold level
Threshold line is located alongwith negative samples orabove some or all positivecurves (S-shaped)
Set the threshold line so that itcrosses only sigmoid curvesof f luorescence accumulationor at the level of betweenfinal values of negative andpositive samples
Drops were not removed fromthe tube walls beforeexperiment started
Curves of f luorescenceaccumulation have negativeor positive steps (Fig. 2)
Select the BaseLineThresholdwindow (the rightmouse button on the graph offluorescence) and indicate
that base line is to becalculated beginning with thefirst cycle after the step
Polymerase (TaqF) was notadded to a reaction mixture
Positive signals are absent forall samples including apositive control
Repeat the experimentbeginning with PCR. Ensureappropriate preparation ofreaction mixtures
Unprocessed (raw) data (Background su bt racted mode).
Fig. 1 Raw curves are normal (FAM) Fig. 2. Baseline has a step:drops werenot removed from the tube walls before theexperiment in the sample indicated as Err
Err
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Processed data
Processed curves are normal; S-shaped; thresholds are located correctly (PCR Base Line
Subtracted Curv e Fit mode)
Fig. 3. FAM channel Fig.4. HEX channel
Fig 5. ROX channelinternal control(-globin gene)
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AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA)
INSTRUMENT
Carry out sample pretreatment and reaction mixture preparation stages according to the
instruction manual. Transparent domed 0.2-mlPCR tubes (detection through the cap of
the tube) are recommended for use.
1. Switch on the instrument and open the Mx3000P program.
2. In the New Experiment Option swindow select Quantitative PCR (Multiple Standards )
and selectTurn lamp on for warm-up.
The lamp should be warmed up for at least 15 min before the experiment starts.
3. Place the tubes in the instrument, secure the locking tool and close the lid.4. Select Opt ics Conf igura tion in the Opt ions menu. In the Dye Assignm ent tab
indicate JOE next to the HEX/JOE fi lter s et option; indicate FAM next to the FAM
filter setoption.
5. In theInst rumentmenu of the Mx3000P program select the Filter Set Gain Sett ings
option. In the opened window set the following parameters of multiplication factors:
Cy5 x4
ROX x1
HEX/JOE x4
FAM x4
Prior to analysis, remove drops from tube walls, because drops falling duringamplification cause signal errors and complicate data analysis. Do not turnstrip/plate over when loading into the PCR instrument.
6. Define parameters of fluorescence acquiring in the Plate Setupmenu. To do this:
a) hold Ctrland use the mouse to select all cells in which test tubes are inserted.
b) define all selected cells as Unknown in the Well type window. In the Collect
f luorescenc e data option select FAM, JOE, ROX, and Cy5. Double click each
cell and enter a sample name (Well Inform at ionwindow). Indicate the positive
control as +, the negative control as . Sample names can be entered during or
after the amplification run in the Plate Setupmenu.
7. Proceed to the Thermal Prof i le Setup tab and set the amplification program (see
Table 7):
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Table 7
DNA HPVtypes 6 and 11 amplification program
for Mx3000P, Mx3005P (Stratagene, USA)
Step Temperature, Time Fluorescencedetection Cyclerepeats
1 95 15 min 1
295 20 s
4560 1 min FAM, HEX/JOE, ROX
This program can be applied for both AmpliSensHPV6/11-FRTandAmpliSensHPV16/18-FRTPCR kits.
Following amplification programs can be used as well:AmpliSens-1 Mx (seeTable 2) and amplification program for HPV HCRDNA types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 9) (this program is applied forAmpliSensHPVHCR screen-titre-FRT PCR kit). AmpliSens-1 Mxamplification program is universal program and it can be used for runningdifferent tests within one run (for example tests for detection of STIs). Fordetection of DNA of HPVHCR and HPV DNA types 6, 11 within one runAmpliSens FRT HR HPVScreenRG4x Program can be applied.
Using amplification program for HPV HCR DNA types 16, 18, 31, 33, 35,39, 45, 51, 52, 56, 58, 59 (from AmpliSensHPVHCR screen-titre-FRT PCRkit) does not affect analytical performances of this PCR kit
Table 8
AmpliSens-1 Mx amplification program
Step Temperature, TimeFluorescence
detection
Cycle
repeats
Segment 1 95 15 min 1
Segment 2(Cycling)
95 5 s
560 20 s
72 15 s
Segment 3
(Cycling)
95 5 s
4060 30 sFAM, HEX/JOE, ROX,
Cy572 15 s
Cy5/Red detection channel should be enabled for running multiplex tests (ifrequired).
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Table 9Amplification program for HPV HCRDNA types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
Step Temperature, TimeFluorescence
detection
Cycle
repeats
Segment 1 95 15 min 1Segment 2 65 2 min 1
Segment 3(Cycling)
95 20 s
564
Touchdown:1 deg. per cycle
25 s
65 55 s
Segment 4(Cycling)
95 20 s
4060 25 s
65 55 sCy5, FAM,
HEX/JOE, ROX
The AmpliSens FRT HR HPV Screen Mx.mxp file enclosed to AmpliSensHPVHCR screen-titre-FRTPCR kitcan be used for programming.
8. In the Plate Setup tab of the Setupmode indicate names of the samples. Define all
samples as Unknown. Select FAM, JOE/HEX, ROX dyes for all samples.
9. Run the program.
10. Proceed to the analysis of results after the end of the run.
Data analysis
1. Proceed to the Analys is item by clicking the button on the toolbar in the Mx300P
program.
2. Ensure that all test samples are enabled (the cells are tinted) in the opened Analys is
Selection/Setuptab. Otherwise, hold Ctrland select all test samples with the mouse.
3. Proceed to the Results tab.
4. On the Dyes shown toolbar activate displaying FAM channel and deactivate
displaying all others channels. Set 150in the Thresho ld f luorescencefield for FAM
channel. One at a time activate next channel and deactivate the previous one and
enter threshold values according to the table:
Cy5 150
ROX 150
HEX/JOE 150
FAM 150
5. Activate displaying all channels (FAM, HEX and ROX buttons should be selected in
the Dyes Show n field at the bottom of the program window).
6. Ensure that the JOE, FAM and ROX fluorescence channels are ticked off in the
Thresho ld f luorescencefield.
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7. Ensure that the threshold line is set correctly. Normally, a threshold line should cross
S-shaped curves of signal accumulation of positive samples and controls and should
not cross the base line. Otherwise, the threshold should be raised.
8. Select Text Report option in the Area to analyzefield. Review the results and export
data for further analysis (for example export to Excel:Fi le>Export Instrument
Data>Export Instrum ent Data to Excel).
TROUBLESHOOTING
Problem Cause How to identify Ways to eliminateDrop of sensitivity dueto deterioration ofprobes
Incorrect storage orutilizing kitcomponents (hightemperature,multiple opening
reagent tubes, foulworking conditions)can cause destroy ofoligonucleotides
Probe destruction canbe find out as a resultof comparison ofexperimental dataobtained at the first
use of the reagentsand after a period oftime or in comparisonwith the reagents ofthe same lot storedproperly. It isappeared as anincrease ofbackgroundfluorescence(fluorescence isassessed in the Rmode at thebeginning of theexperiment) twice asmuch in differentexperiments (in casethe same instrumentis used). Note!Effectof f luorescenceincrease can appearafter cleansing theoptical system
Use mixtures storedunder statedconditions untillabeled expire date(see part Stability
and Storage in theInstruction Manual)
Drop of sensitivity dueto decrease ofpolymerase (TaqF)activity
Incorrect storage orcontamination causeenzymedeterioration
It appears as a fail ofthe positive control orif a boundary Ct valueof the positive controlis greater than thethreshold of weaksamples
Use the reagentstored under requiredcondition (see partStability and Storagein the InstructionManual) or use a newreagent
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Problem Feature Ways to eliminate
Contamination of a specificDNA
Detection of a signal for anegative control in anychannel
Repeat the experiment. Takemeasures to detect andeliminate the source ofcontamination
The volume of a DNA sampleadded to a reaction tube isless than required/DNAsamples is not added
Background signal of asample is much stronger thanthe others (see raw curvesR-mult icompon ent viewmode) The sample is negative
Repeat PCR for this sample
The volume of a reactionmixture added to a reactiontube is less thanrequired/reaction mixture isnot addedorthe volume of aDNA sample added to areaction tube is greater than
required
Background signal of asample is much lower than theothers (see raw curves-R-mul t icompo nent v iewmode)
If the sample is negative,repeat the test
Incorrect adjustment of thethreshold level
Threshold line is located alongwith negative samples orabove some or all positivecurves (S-shaped)
Set the threshold line so that itcrosses only sigmoid curvesof f luorescence accumulationor at the level of betweenfinal values of negative andpositive samples
Polymerase (TaqF) was notadded to a reaction mixture
Positive signals are absent forall samples including apositive control
Repeat the experimentbeginning with PCR. Ensureappropriate preparation ofreaction mixtures
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REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 21 of 22
Unprocessed data(R mode)
Fig. 1. Raw curves are normal Fig. 2. Level of background signal pointon a possible problem: DNA samplewas not added to the Err tube
Processed data
Normal curves that were processed, dRmode (S-shaped)
Fig. 3. FAM channel
Fig. 4. HEX channel
Fig. 5. ROX channelinternal control
(-globin gene)
Err
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List of Changes Made in the Instruction Manual
VERLocation of
changesEssence of changes
02.04.14
SA
Cover page
Footer
Address of European representative was added
REF R-V11(RG,iQ,Mx)--B was deleted