Introduction to Real Time PCR for Warwick Users
There is an ABI 7000 Sequence Detection System in D126 which anyone can book on line. Please
note that our instrument is not the ‘Fast’ Real-Time PCR platform. These notes are to help you set up
Real Time PCR assays for gene expression for the first time. Refer also to the MIQE guidelines (pdf on
our website).
Planning your Assays
1. Reverse Transcription
To measure gene expression, RNA needs to be transcribed into single-stranded cDNA, then it is
ready for the PCR reaction; there is no need for a separate second strand synthesis. Think about
whether everything you intend to assay is polyadenylated, in which case use an Oligo dT primer,
otherwise use random hexamers, or in some cases a gene-specific primer. Note that eukaryotic 18S
ribosomal RNA, frequently used as an endogenous control, is not polyadenylated, and neither is
prokaryotic message. Remember to set up a ‘no RT’control for each template, which controls for
contaminating genomic DNA and is especially important if your amplicon doesn’t span an exon:exon
boundary (e.g., prokaryotic assays).
2. Real Time Chemistry
‘Taqman’ chemistry is more sensitive and more specific but SYBR Green is cheaper. There is more
information about these chemistries through links on our website. Taqman assays can be bought ‘off
the shelf’, or designed through ABI’s website. SYBR Green primers need to be designed using the
appropriate software (Primer Express software is recommended). It is critical to design the smallest
amplicon possible – between 60 and 150 bp is optimal. You should consider serial dilution curves to
make sure you are working in the linear range of the PCR, and that the reaction is efficient.
Alternatively, Primer Design can design bespoke, validated assays for you for either chemistry at a
competitive price.
3. Endogenous Controls
Gene expression assays require a control gene to be run in parallel which does not fluctuate across
the conditions you are investigating. Many endogenous controls are available as off-shelf-assays
from ABI. The control gene should really be validated in your experimental conditions and you may
need to look at several candidates for endogenous controls. If you plan to multiplex your
endogenous control with your gene of interest, both assays need to be optimised such that the
efficiency of neither PCR reaction is compromised by multiplexing. Again, Primer Design can advise
and design endogenous controls.
4. Plate Set Up
Plates should be set up in your own lab, and only sealed plates brought into D127 for running on the
instrument. Ideally you need clean lab space away from areas where similar templates have already
been amplified and manipulated – Real Time PCR is a sensitive technique which is easy to
contaminate. Filter tips are advised. Samples and negative controls should always be run in
triplicate. Triplicates should be placed in horizontal rows across the plate as this makes the data
easier to analyse in Excel. Any number of amplicons can be run on one plate (provided they were
designed to have the same annealing temperature), and it is not necessary to run a whole plate.
5. Running a Plate on the 7000
Before you use the instrument for the first time, carefully read the instructions at the end of this
document. Booking sheets are available on line. The instrument must be switched off at the end of
your run, and it is recommended that the computer is restarted before every run. Note that this
computer is disconnected from the network, and please bring a memory stick to back up your data
after every run. Often the log on profile becomes unstable, and data can be lost.
6. Analysing your data
User Bulletin #2 (downloadable from our website) gives a good overview of various methods for
analysis. The software for analysing your data is available via the Novell network: Life Sciences >
Prism> ABI Prism 7000-v1.2.3 (ABI Prism 7000 is older software for older data).
Ordering Consumables
The following compatible plastics are available in stores:
(Starlab)
Semi skirted 96 well Plate compatible with ABI 7000 I1402-9800-C Pack size: 10
(Alphalabs)
RT 50μm Sealing films compatible with ABI7000 LW2786 pack size: 100
Other plastic ware is available
Master mix kits are available from various suppliers.
Running a Plate on the 7000 Sequence Detection System
1. Switch on the laptop computer.
2. If a plate centrifuge is available, it is a good idea to give your plate a quick pulse before running.
3. When the login window opens, check the label on the laptop for logon details. The profile has
been unstable in the past and so may change. Log on as ‘workstation only’. Ensure that the network
cable is not connected.
4. Once the computer has started up you can then switch on the instrument (power button on the
front).
5. First time users: Go to ‘NewRealTime’s Documents’ on the desktop and open ‘SDS files’. Make a
new folder using your own stores code. All of your data will be stored here, but you are responsible
for backing it up yourself.
6. Double click the ‘7000 Software’ icon on the desktop.
7. File > new.
8. Select the Assay type from the drop down list – ‘Absolute Quantification’ for SYBR green, ‘Relative
(ddCt) Quantification Plate’ for TaqMan assays. Leave the defaults for ‘container’ and ‘template’,
then click Next
9. Select detector - or make a new one as appropriate for new assays. If this is the first time you’ve
used this instrument you should set up a new detector for each amplicon you will be running. These
will remain in the list for you to select in future. Make sure you choose the correct fluorophore –
SYBR for SYBR green assays, FAM (with no quencher) for TaqMan assays. ‘Add’ to your plate
document and click Next. You can also make and add detectors in the Well Inspector.
10. Click Finish.
11. In the ‘set up’ tab highlight triplicate sample wells, then go to> View >Well Inspector to enter the
sample and assay details for each set of wells.
12. Type in the sample name and select detector (check left hand box). Always leave the passive
reference as ROX. For Absolute Quantification (SYBR Green) assays, you are able to define the task
for each triplicate set. ‘Unknown’ for samples under test, ‘NTC’ for negative (no template) controls,
‘Standard’ for points of a dilution curve, if the quantity is entered the software will plot the curve.
When the plate layout is complete, close the Well Inspector.
13. Click away from the ‘Setup’ tab to the ‘Instrument’ tab. Provided that your primers (and probe)
were designed using ABI software, or purchased from ABI, there is no need to change any of the PCR
cycling conditions.
14. Insert volume of sample (in μL) used into the Sample Volume field box – usually 20μL.
15. Uncheck the ‘9600 Emulation’ box.
16. Select Add Dissociation Stage for all Absolute Quantification (SYBR Green) assays (you cannot
add this stage if you are setting up a Relative Quantification Plate using Taqman assays).
17. Then save the file in your stores code folder in ‘SDS files’ (> File > Save As).
18. Place your plate into the instrument ensuring that the black precision plate holder is in place and
that you have placed a compression pad on top of your plate.
19. Close the instrument door.
20. Click Start Button on the instrument control tab.
21. Listen for the ‘Whirr clunk’ sounds from the instrument to ensure that it is running and
communicating with the laptop.
22. Your results will automatically be saved to your folder when the run has finished. The progress of
your run can be viewed by selecting ‘Amplification plot’ in the ‘Results’ tab.
23. Ensure you copy your data on to a memory stick as soon as the run as finished. The user profile
has been unstable in the past and documents get lost.
24. To export data from an ‘Absolute Quantification’ (SYBR Green) run, go to File > Export> Ct… and
save the file in your folder. The csv file can be opened in Excel.
25. To export data from a ‘Relative Quantification (ddCt) Plate’ (Taqman) first open the plate in a
‘Relative Quantification (ddCt) Study’, and either analyse here, or click Finish and go to File > New >
Export > Ct…and save the file in your folder. The csv file can be opened in Excel.
26. Close SDS Software when the run is completed, switch off the instrument and shutdown the
computer. If you are going to run another plate, still do a full reboot of the instrument and laptop to
avoid stability issues.