Introduction to the Rotor-Gene 3000

1
Getting Started on the Rotor-Gene-6000
Jennifer McMahon, PhD
www.corbettresearch.com
2
the worlds only real-time rotary thermo-optical
analyser
3
Cross-section of rotary optics
4
Getting Started
?Click on Rotor-Gene icon
Click on advanced tab for step by step run set up
? Choose run template depending on
chemistry Dual labeled probes hydrolysis
probes SYBR intercalating dyes
5
Select the rotor you are using, put the rotor
into the machine with the locking ring
attached The locking ring tick box must be ticked
to proceed Click Next
6
Insert user name, any notes (primer conc, primer
sequence mix used etc) and volume required
(10-25uL) This info is saved and can be printed
in reports Click Next
7
?click on Edit Profile to change run parameters
section to be altered is greyed out, click on
Hold Temperature to change temperature, click
on Hold Time to change time ? Always read
manufacturers instructions regarding Hot start
Taq activation times
8
Click on Cycling to adjust the cycling
parameters Default cycle number is 40 To change
a step click on the temperature and adjust then
click on the time and adjust
9
Data is acquired each cycle (blue dots) No blue
dots no data very bad Acquire at the end of
the extension phase Click on Aquiring to Cycling
A to select colours Move the colours you want
from Available Channels to Acquiring Channels
using arrows Use Green channel for SYBR green
10
With SYBR use Melt to look for primer-dimer or
non-specific product Melt should start from
acquiring temperature If you start the melt from
annealing temp the melt may have a shoulder on
it Use default settings, make sure you are
acquiring data (blue dots)
11

Gain can be compared to the aperture on a camera
and is used to fit data on raw scale If your
starting fluorescence signal is weak the gain
should be high, if strong the gain should be
low Many people just accept default gain and go
to Next on this screen, others optimise the
gain for each run Click on Gain Optimisation to
begin
12
Click on Optimise Acquiring and Perform
Calibration Before 1st Acquisition If you have
different colours in different positions click
Edit The colour you want to use must be in the
tube position for calibration to be
successful Dont set gain on no template
controls
13
Click on Start Run
Save file name in directory of your
choice ? Default nomenclature has name of
template file, date and version (1 for 1st run
that day etc)
14
To save as a template open template folder
C\Program Files\Rotor-Gene 6000
Software\Templates and save as template file
(.ret) Run files are .rex
15
?Choose to enter sample names now or click
Finish and enter sample names later
Run will commence, while the experiment is
running you will see Raw Channel - fluorescence
collected cycle by cycle Temperature - profile
as temperature cycles Profile Progress tells
you time remaining, shows you where you are.
Skip allows you to skip to next step (e.g. from
cycling to melt) Add 5 cycles lets you add
cycles at the end
16
Adding sample names and types
Can highlight blocks of cells for naming etc
(like excel) Samples with the same name will be
treated as replicates regardless of position Type
can be standard, no template control, unknown
(sample)
Click on Edit Samples on RHS of screen
17
Drop down Type menu to view options If you
choose Standard you have to add a value for
concentration If you are running two standards
define one set on page 1 then make a new page
(page 2) to define the second set of
standards Groups can be used to define e.g HKG
(housekeeper) and GOI (gene of interest
18
Can choose colour from palette and can set colour
gradient across range of samples/standards
19
Post run analysis - basic
Raw data is displayed on screen during the run
and at the end of the run. Analysis is needed to
correct for background etc
Click on Analysis on the main toolbar at the
top Click on Quantitation tab Click on channel
so that it gets greyed out Click on Show
20
The software looks at the standards and draws a
threshold, calculates the standard curve and the
replicates
21
Dynamic Tube normalisation should be on to
correct for background Slope Correct should be
on for dual labeled probes Ignore first can be
useful if the first few cycles are different
(usually higher) than the rest of the run
22
Dynamic Tube Normalisation
This option is ticked by default and is used to
determine the average background of each
individual sample just before amplification
commences. Standard Normalization simply takes
the first five cycles and uses this as an
indicator for the 'background' level of each
sample. All data points for the sample are then
divided by this value to normalize the data. This
process is then repeated for all samples. This
can be inaccurate as for some samples the
background level over the first five cycles may
not be indicative of the background level just
prior to amplification. Dynamic Tube
Normalization uses the second derivative of each
sample trace to determine a starting point for
each sample. The background level is then
averaged from cycle 1 up to this starting cycle
number for each sample. This method gives the
most precise quantitation results. Alternatively
with some data sets it may be necessary to
disable the dynamic tube normalization. If this
is the case the average background for each of
the samples is only calculated over the first 5
cycles. If the background is not constant over
the cycles before amplification it will result in
less precise data.
Slope correct
The background fluorescence (Fl) of a sample
should ideally remain constant before
amplification. However, sometimes the Fl-level
can show an increase or decrease due to the
effect of the chemistry being run and produce a
skewed noise level. The Noise Slope Correction
option uses a line-of-best-fit to determine the
noise level instead of an average, and normalizes
to that instead. Turning on this option can
tighten replicates if your sample baselines are
noticeably sloped. This function improves the
data when raw data backgrounds are seen to slope
upward or downward before the amplification
Takeoff point (CT). It is very helpful for runs
when for example the FAM background is seen to
creep upwards due to gradual probe
autohydrolysis.
Ignore first
The first couple of cycles in a quantitation run
are not usually representative of the rest of the
run. For this reason, you may get better results
if you select to ignore the first few cycles. If
the first cycles look similar to cycles after
them, you will gain better results by disabling
this function, as the normalization algorithm
will have more data to work with. You can ignore
up to ten cycles.
23
If there are no standards in the run you have to
set the threshold manually. Click on Threshold
button Click to select the threshold height
comes up, move the line up or down to select the
threshold Do this in log scale
Linear scale view, click on log scale button to
get log scale
Log scale view, click on linear scale button to
get linear scale
24
Standards are plotted in blue, samples as
red Software calculates relationship value (R2)
ideally 0.99 Software calculates the efficiency
out of 1 1 means 100 efficient or doubling
every cycle
25
Software calculates Rep. Ct to get replicate Ct
from triplicates (geometric mean) Software
calculates Rep. Ct Stc to get standard
deviation for triplicates Software calculates
Rep. Calc Conc. to calculate concentration from
a standard curve Columns can be switched off by
right clicking on grey bar and changing
options Table can be exported to MS Excel by
right clicking on table text and choosing export
to excel
26
Raw melt data can be viewed at end of run.
Fluorescence decreases as temperature
increases The software plots a mathematical
derivative to turn the raw data into a peak Click
on Analysis then highlight Melt then click
Show