Standard Operating Procedure
Title: Gene Expression Analysis by Quantitative Real-Time PCR
Department: Agronomy
Created by: Jason W. Haegele
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr. Mark Westgate
Lab Supervisor: Maria Hartt
Date approved: 21 March 2008
Procedure Overview: This procedure describes comparative gene expression analysis using
real-time PCR. Reverse-transcribed RNA (cDNA) is used as the template for amplification.
The Stratagene Mx4000 real-time PCR instrument in the Iowa State University DNA Facility
is used. Real-time PCR is an extremely useful, yet complex research tool. It is advised that
the user of this procedure thoroughly understand the principles and limitation of real-time
PCR before proceeding. Contact Jon Mlocek (jmlocek@iastate.edu) for training on the
Stratagene Mx4000 real-time PCR instrument.
Equipment and reagents necessary:
Reagents:
cDNA stored at -20oC
Brilliant II SYBR Green QPCR Master Mix (Stratagene: 600828), 1 kit per 400 reactions
Kit contains:
2X Brilliant II SYBR Green QPCR Master Mix
Reference Dye, 1 mM
Forward primer (IDT), see appendix, 0.8 l per reaction
Reverse primer (IDT), see appendix, 0.8 l per reaction
Alien QRT-PCR Inhibitor Alert (Stratagene: 300600), 1 kit per 400 reactions
Kit contains:
Alien RNA transcript
Alien Primer Mix, 2.5 M
Deionized water
Miscellaneous:
Micropipettors and tips, 10 l and 1 ml
1.5 ml microcentrifuge tubes, 1 for dye dilution and 1 per primer pair
Vortex
Mx4000 96-well plates, (Stratagene: 410021)
Centrifuge, 1190 Molecular Biology Building
Stratagene Mx4000 real-time PCR instrument, 1190 Molecular Biology Building
Procedure:
1. Dilute the reference dye: The kit includes 100 l of 1mM reference dye. This dye must
be diluted 1:500 before further use. Use a pipette to transfer 2 l of dye to an
appropriately labeled 1.5 ml microcentrifuge tube. Use a pipette to add 998 l of
deionized water. Vortex gently to mix. Store this diluted dye at 4oC for future use.
2. Primer preparation: See standard operating procedure ‘Preliminary Primer Testing for
Quantitative Real-Time PCR’ for instructions on preparing primer solutions.
3. Prepare the PCR mix: It is advisable to run one plate per pair of forward/reverse primers
to simplify set-up. Determine the number of samples (plus controls and standards) that
will be run simultaneously on one plate. Multiply this number by the following volumes
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and use a pipette to transfer the resulting volumes in order shown to an appropriately
labelled 1.5 ml microcentrifuge tube. The total reaction volume including template cDNA
is 20 l.
Component
Amount
Deionized water
7.52l
Brilliant II SYBR Green QPCR Master Mix
10 l
5 M forward primer
0.8 l
5 M reverse primer
0.8 l
Diluted reference dye
0.38 l
4. Gently mix the PCR mix by inversion. Do not vortex as this may create bubbles which
will cause problems for the real-time PCR data acquisition.
5. Use a pipette to transfer 19.5 l of the PCR mix to each well in the real-time PCR plate.
6. Use a pipette to transfer 0.5 l of template cDNA to each well. Place the template on the
side of the well and not directly into the PCR mix. Document the location of each
template.
7. During the reverse transcription step (see ‘Reverse Transcription of RNA for Quantitative
Real-Time PCR’) an external control (‘Alien RNA Transcript’) was added to each sample.
Separate replicated reactions will be run for each sample using the ‘Alien RNA
Transcript’ primers instead of gene specific primers. Determine the number of external
control amplification reactions that will be run simultaneously on one plate. Multiply this
number by the following volumes and use a pipette to transfer the resulting volumes in
order shown to an appropriately labelled 1.5 ml microcentrifuge tube. The total reaction
volume including template cDNA is 20 l.
Component
Amount
Deionized water
8.32l
Brilliant II SYBR Green QPCR Master Mix
10 l
Alien Primer Mix
0.8 l
Diluted reference dye
0.38 l
8. Gently mix the external control reaction PCR mix by inversion. Do not vortex as this
may create bubbles which will cause problems for the real-time PCR data acquisition.
9. Use a pipette to transfer 19.5 l of the external control reaction PCR mix to the
designated wells in the real-time PCR plate.
10. Use a pipette to transfer 0.5 l of template cDNA to each external control reaction well.
Place the template on the side of the well and not directly into the PCR mix. Document
the location of each template.
11. Place the accompanying lid on the PCR plate and place the plate in a cooler or
Styrofoam box for transport the DNA facility. The transport container should contain a
small amount of ice to keep the plate contents cold.
12. Once at the DNA facility, use the centrifuge there to briefly centrifuge the plate to spin
down the contents. Setting #5 should be used. Consult the facility staff for instructions
on operation.
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13. Begin the PCR operation as instructed during the initial training. Place the PCR plate in
the instrument when instructed by the computer. Specifically, the operating conditions
are as follows.
Cycles
Duration of Cycle
Temperature
1
10 minutes
95oC
40
30 seconds
95oC
60 seconds
60oC
14. Bring the PCR plate back to 1514 Agronomy Hall and place in an appropriately labeled
container for EH&S disposal.
Personal Protective Equipment / Engineering Controls:
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
Handling & Storage Precautions:
Brilliant II SYBR Green QPCR Master Mix: Store at -20oC upon receipt in an appropriately
labelled container. Once the master mix is thawed, store at 4oC in an appropriately
labelled container.
Alien QRT-PCR Inhibitor Alert: Store at -80oC in an appropriately labelled container.
Waste Disposal Procedures:
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess
solutions) must be placed in an appropriately labeled hazardous waste container for EH&S
disposal. Compatible substances may be combined into one waste container.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Brilliant II SYBR Green QPCR Master Mix: Soak up with an inert absorbent material.
Alien QRT-PCR Inhibitor Alert: Soak up with an inert absorbent material.
Health & Safety Summary for Required Reagents:

The Brilliant II SYBR Green QPCR Master Mix emits toxic fumes when heated above its
boiling point; however, these conditions are unlikely to be met during the conditions of this
protocol.

There is no MSDS available for the Alien QRT-PCR Inhibitor Alert Kit. This kit contains
RNA and associated primers so there should not be any significant health or safety risks
associated with these materials.
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Chemical name
Brilliant II SYBR Green
QPCR Master Mix
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Incompatibilities
Acid chlorides,
phosphorus halide, strong
acids, strong oxidizing
agents, strong reducing
agents. Sensitive to
moisture.
 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read and understand the
contents of the entire MSDS(s) before starting this procedure.
References:
2007. Quantitative RT-PCR Protocol (SYBR Green I). Schnable Lab (Iowa State University).
Available online at:
http://schnablelab.plantgenomics.iastate.edu/docs/resources/protocols/pdf/realtime_RTPCR.2007.04.01.pdf. Last accessed: 19 March 2008.
Product manual for Brilliant II SYBR Green QPCR Master Mix.
Methods and Applications Guide: Introduction to Quantitative PCR.
Stratagene website.
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Appendix: Primer Sequences
Gene:
Forward Primer:
Reverse Primer:
Approximate product
size (base pair):
cyPPDK1
TAAACGATGCGGAGAAGCTCGTGA
TAACATCATCCACCCAGGCCATGA
168
ZmSPK1
CATCGCTGCTGCATTCAAAGCCTA
ACCGACCAGCATCACGTAAAGAGT
143
Sh2
TATAGATCGGCTGCGTTTGCGTCT
AGCGGCTCTTACCATACCAAGGTT
91
Sh1
TCAATGCCTCCTTTCCTCGTCCTT
AAGCTCACCGTGCCCTTGTAGTTA
160
Wx1
TCGCGTCCTGCTGGTTCATTATCT
TCATCCAGTTGATCTTCCGGCCTT
84
O2
ACAATCACACTGGAGGTAGCAGCA
GCCTGCAGTTTGAGCGTGGTTATT
101
Dbf1
CAAGAGCAAGGCGATGCCAATCAA
ACGGAACCTCGCTGAAATCCAACT
191
Dhn1
ATGTGACAGGGACAGGGACAGTTT
AGCCACTCGCAAGTGCTGTACTAT
128
15-kD beta-zein
ATGATGATGGCGCAGAACATGC
AATCAGTAGTAGGGCGGAATGGCA
113
27-kD gamma-zein
TATGTGCTGTAGTATAGCCGCTGG
ATTGCTCACACTGACACTGCCAC
112
19-kD alpha-zein B1
TTGCCTCCTTATGCTCCTTGGTCT
AAGGTAAGATGCCAGCTGCGATTG
188
ZmACO20
CTCATCCTGCTGCTCCAGGACGAC
TCCACGATACACGCATAACCACCGT
?
Acc1
TCCCAACTCTTGCTTGGAGTGGAT
TGCAACTGCTTCCTCTGTGGTAGT
119
beta-tubulin
ACCAGATCGGCGCCAAGTTCT
CATCATGTTCTTGGCATCCCACA
?
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