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TABLE OF CONTENTS
INTENDED USE .................................................................................................................. 3
AMPLIFICATION AND DATA ANALYSIS WITH Rotor-Gene 3000/6000 (Corbett
Research, Australia) INSTRUMENTS ................................................................................. 3
AMPLIFICATION AND DATA ANALYSIS WITH iCycler iQ5 (Bio-Rad, USA)
INSTRUMENTS................................................................................................................. 11
AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA)
INSTRUMENT ................................................................................................................... 16
INTENDED USE
Guidelines describe the procedure of the use of AmpliSens HPV 6/11-FRT PCR kit for
qualitative detection and differentiation of types 6 and 11 of human papillomavirus (HPV)
DNA by means of real-time hybridization-fluorescence detection with the following realtime 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-ml PCR 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.
When working 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 (not
empty).
3. Program the instrument to run amplification and detection:
In the opened window select Advanced mode. Activate the New button.
Select 36-Well Rotor (or 72-Well Rotor) and No Domed Tubes/Locking ring
attached. Click Next.
In the opened window define the operator and set the reaction volume: Reaction
volume is 25 l. Tick off the 15 l oil layer volume option.
set amplification program.
REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 3 of 22
Table 1
DNA HPV types 6 and 11 amplification program for
Rotor-Gene 3000/6000 (Corbett Research, Australia)
Stage
emperature,
Time
Hold
95
95
15 min
15 s
60
35 s
Cycling
Fluorescence
detection
FAM/Green,
JOE/Yellow,
ROX/Orange
Cycle
repeats
1
45
This program can be applied for both AmpliSens HPV 6/11-FRT and
AmpliSens HPV 16/18-FRT PCR kits.
Following amplification programs can be used as well: AmpliSens-1 RG (see
Table 2) and amplification program for HPV HCR DNA types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 3) (this program is applied for
AmpliSens HPV HCR screen-titre-FRT PCR kit). AmpliSens-1 RG
amplification program is a universal program and it can be used for running
different tests within one run (for example tests for detection of STIs). For
detection of DNA of HCR HPV and HPV DNA types 6, 11 within one run
AmpliSens FRT HR HPV ScreenRG4x 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 AmpliSens HPV HCR screen-titre-FRT PCR
kit) does not affect analytical performances of this PCR kit.
Table 2
AmpliSens-1 RG amplification program
Step
Temperature,
Time
Hold
Cycling 1
95
95
60
72
95
15 min
5s
20 s
15 s
5s
Cycling 2
60
20 s
72
15 s
Fluorescence
detection
FAM/Green,
JOE/Yellow,
ROX/Orange,
Cy5/Red
Cycle
repeats
1
5
40
Cy5/Red detection channel should be enabled for running multiplex tests (if
required).
Table 3
Amplification program for HPV HCR DNA types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
Step
Temperature,
Time
Hold
Hold 2
95
65
95
64
Touchdown:
1 deg. per cycle
65
95
60
15 min
2 min
20 s
Fluorescence
detection
25 s
55 s
15 s
25 s
65
25 s
FAM/Green,
JOE/Yellow,
ROX/Orange,
Cy5/Red
Cycling
Cycling 2
Cycle repeats
1
1
40
of the Name field. Click New. Third page for information about samples will appear.
Indicate Human (ROX/Orange channel) in the Page area of the Name field.
Data analysis
Review row data in all three channels (FAM/Green, JOE/Yellow and ROX/Orange) by
selecting Cycling A buttons on the Channels bar. Pay attention to the followings:
among all signals detected in the channel at least one should be positive (a positive
signal has typical S-shape, see Fig.1). If the experiment is correct, the signal of
positive control C+ must be detected.
If positive signals were not detected in a channel the result of data analysis can
be incorrect.
If background signal of some samples greatly differs from all other samples it indicates
incorrect dispensing reaction mixture or DNA sample during tube preparation (see
Fig. 2a).
1. Make sure that all analyzed samples are activated on the legend at the right.
2. Activate the Analysis button then select the Quantitation mode. Activate the Cycling
A. FAM/Cycling A. Green button; click Show.
3. Set Threshold = 0.03.
4. Select Linear scale. Activate the Dynamic tube and Slope Correct buttons in the
main window menu (Quantitation analysis).
5. Select the More Settings/Outlier Removal parameter and set the NTC threshold
value as 10 %.
6. Carry out data analysis for JOE/Yellow and ROX/Orange channels similarly to that for
FAM/Green channel.
In some rare cases due to descending character of the curves at the beginning
of the experiment, fluorescent curves can cross the threshold line at first cycles
and Rotor-Gene program can accept these results as positive (Fig. 7). Use
Eliminate cycles before option and enter 5.
7. For data analysis select Analysis then Quantitation.
TROUBLESHOOTING
Review this section before running the experiment
Problem
Cause
How to identify
Negative samples are
analyzed as positive
samples by RotorGene program
Incorrect
mathematical
processing of
negative samples in
the presence of drop
of fluorescence at
the initial cycles
(Fig. 2a)
Lenses
contamination leads
to reduction of
effectiveness of
fluorescence
excitation and
detection. Primarily
this has an impact
on the samples
containing small
quantity of specific
DNA which show
low fluorescence
growth
Incorrect storage or
utilizing kit
components (high
temperature,
multiple opening
reagent tubes, foul
working conditions)
can cause
oligonucleotides
destruction
Ways to eliminate
A normal positive
sample has a Sshaped curve of
fluorescence
accumulation (Fig. 1,
3-5). The negative
samples processed
incorrectly appear as
quite straight bottomup lines (Fig. 7)
The red threshold line
crosses (or touches)
fluorescence curves
at the left (initial
cycles) on the chart of
processed
fluorescence curves
(Fig. 6)
Low background
signal in all four
detection channels
(<1) along with
maximum value of the
gain parameter (10)
Desensitization due to
decrease of
polymerase (TaqF)
activity
Incorrect storage or
contamination cause
enzyme
deterioration
Feature
Detection of a signal of the
negative control in any
channel
Background signal of a
sample is much stronger than
the others (see raw curves,
Fig. 2b). The sample is
negative
Background signal of a
If the sample is negative,
sample is much lower than the repeat the test
others (see raw curves, Fig.
2b).
Ways to eliminate
Repeat the experiment. Take
measures to detect and
eliminate the source of
contamination
Repeat PCR for this sample
Temperature,
Time
95
95
60
15 min
20 s
1 min
Fluorescence
detection
Cycle
repeats
1
45
This program can be applied for both AmpliSens HPV 6/11-FRT and
AmpliSens HPV 16/18-FRT PCR kits.
Following amplification programs can be used as well: AmpliSens-1 iQ (see
Table 5) and amplification program for HPV HCR DNA types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 6) (this program is applied for
AmpliSens HPV HCR screen-titre-FRT PCR kit). AmpliSens-1 iQ amplification
program is universal program and it can be used for running different tests
within one run (for example tests for detection of STIs). For detection of DNA
of HCR HPV and HPV DNA types 6, 11 within one run AmpliSens FRT HR
HPV ScreenRG4x 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 AmpliSens HPV HCR screen-titre-FRT PCR
kit) does not affect analytical performances of this PCR kit
Table 5
AmpliSens-1 iQ amplification program
Step
Temperature,
Time
95
95
60
72
95
60
72
15 min
5s
20 s
15 s
5s
30 s
15 s
Fluorescence
detection
Cycle
repeats
1
5
40
Cy5/Red detection channel should be enabled for running multiplex tests (if
required).
Table 6
Amplification program for HPV HCR DNA types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
15 min
15 s
Fluorescence
detection
Cycle
repeats
1
55 s
25 s
15 s
55 s
25 s
Real-time
Step
Temperature,
Time
Cycle 1
95
95
65
Touchdown:
1 deg. per cycle
65
95
60
65
Cycle 3
Cycle 4
41
Create a new plate setup in the Edit Plate Setup tab. Indicate samples as Unknown.
Proceed to the Select fluorofores tab and set fluorescence detection for all tubes in the
FAM, HEX and ROX channels. Go back to the Samples: Whole plate loading and click
the Per Dye Layer button.
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 the PCR Quantification tab in the Data Analysis mode.
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 Defined, 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 Results button (below the fluorofore buttons).
5. Proceed to the PCR Standard Curve tab, review parameters of the reaction, copy the
results.
6. Click on the results grid using the mouse right button. Select Export to Excel from 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
REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 12 of 22
data analysis). Results in the FAM, HEX, ROX, and then Cy5 channels follow
consistently.
TROUBLESHOOTING
Cause
How to identify
Problem
Incorrect storage or
utilizing kit
components (high
temperature,
multiple opening
reagent tubes, foul
working conditions)
can cause destroy of
oligonucleotides
Incorrect storage or
contamination cause
enzyme
deterioration
Problem
Contamination of a specific
DNA
Feature
Detection of a signal for a
negative control in any
channel
Ways to eliminate
Use mixtures stored
under stated
conditions until
labeled expire date
(see part Stability
and Storage in the
Instruction Manual)
Ways to eliminate
Repeat the experiment. Take
measures to detect and
eliminate the source of
contamination
Repeat PCR for this sample
Curves of fluorescence
accumulation have negative
or positive steps (Fig. 2)
Err
Processed data
Processed curves are normal; S-shaped; thresholds are located correctly (PCR Base Line
Subtracted Curve Fit mode)
x4
ROX
x1
HEX/JOE
x4
FAM
x4
Prior to analysis, remove drops from tube walls, because drops falling during
amplification cause signal errors and complicate data analysis. Do not turn
strip/plate over when loading into the PCR instrument.
6. Define parameters of fluorescence acquiring in the Plate Setup menu. To do this:
a) hold Ctrl and 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
fluorescence data option select FAM, JOE, ROX, and Cy5. Double click each
cell and enter a sample name (Well Information window). Indicate the positive
control as +, the negative control as . Sample names can be entered during or
after the amplification run in the Plate Setup menu.
7. Proceed to the Thermal Profile Setup tab and set the amplification program (see
Table 7):
Table 7
DNA HPV types 6 and 11 amplification program
for Mx3000P, Mx3005P (Stratagene, USA)
Step
Temperature,
Time
95
95
60
15 min
20 s
1 min
Fluorescence
detection
Cycle
repeats
1
45
This program can be applied for both AmpliSens HPV 6/11-FRT and
AmpliSens HPV 16/18-FRT PCR kits.
Following amplification programs can be used as well: AmpliSens-1 Mx (see
Table 2) and amplification program for HPV HCR DNA types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 9) (this program is applied for
AmpliSens HPV HCR screen-titre-FRT PCR kit). AmpliSens-1 Mx
amplification program is universal program and it can be used for running
different tests within one run (for example tests for detection of STIs). For
detection of DNA of HPV HCR and HPV DNA types 6, 11 within one run
AmpliSens FRT HR HPV ScreenRG4x 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 AmpliSens HPV HCR screen-titre-FRT PCR
kit) does not affect analytical performances of this PCR kit
Table 8
AmpliSens-1 Mx amplification program
Step
Temperature,
Time
Segment 1
Segment 2
(Cycling)
95
95
60
72
95
15 min
5s
20 s
15 s
5s
Segment 3
(Cycling)
60
30 s
72
15 s
Fluorescence
detection
Cycle
repeats
1
5
40
Cy5/Red detection channel should be enabled for running multiplex tests (if
required).
Table 9
Amplification program for HPV HCR DNA types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
15 min
2 min
20 s
Fluorescence
detection
Cycle
repeats
1
1
25 s
55 s
20 s
25 s
Cy5, FAM,
HEX/JOE, ROX
Step
Temperature,
Time
Segment 1
Segment 2
95
65
95
64
Touchdown:
1 deg. per cycle
65
95
60
65
55 s
Segment 3
(Cycling)
Segment 4
(Cycling)
40
150
ROX
150
HEX/JOE
150
FAM
150
5. Activate displaying all channels (FAM, HEX and ROX buttons should be selected in
the Dyes Shown field at the bottom of the program window).
6. Ensure that the JOE, FAM and ROX fluorescence channels are ticked off in the
Threshold fluorescence field.
REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 18 of 22
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 analyze field. Review the results and export
data for further analysis (for example export to Excel: File>Export Instrument
Data>Export Instrument Data to Excel).
TROUBLESHOOTING
Cause
How to identify
Problem
Incorrect storage or
utilizing kit
components (high
temperature,
multiple opening
reagent tubes, foul
working conditions)
can cause destroy of
oligonucleotides
Incorrect storage or
contamination cause
enzyme
deterioration
Ways to eliminate
Use mixtures stored
under stated
conditions until
labeled expire date
(see part Stability
and Storage in the
Instruction Manual)
Problem
Contamination of a specific
DNA
Feature
Detection of a signal for a
negative control in any
channel
Background signal of a
sample is much stronger than
the others (see raw curves
R-multicomponent view
mode) The sample is negative
Background signal of a
If the sample is negative,
sample is much lower than the repeat the test
others (see raw curves - Rmulticomponent view mode)
Ways to eliminate
Repeat the experiment. Take
measures to detect and
eliminate the source of
contamination
Repeat PCR for this sample
Err
Processed data
Normal curves that were processed, dR mode (S-shaped)
Location of
changes
Cover page
Footer
Essence of changes
Address of European representative was added
REF R-V11(RG,iQ,Mx)--B was deleted