This document describes PrimeTime® qPCR products for gene expression analysis, including probe- and primer-based assays for human, mouse, and rat sequences as well as custom assays. It provides details on master mixes, probes, controls, and an assay design and ordering process to ensure specific and efficient assays. The assays are guaranteed to have high efficiency and sensitivity for accurate quantification of gene expression.

1. PrimeTime® qPCR products
for gene expression
1

2. • Assays—probe- and primer-based
• PrimeTime® qPCR Assays
(for human, mouse, and rat)
• PrimeTime® Custom qPCR Assays
(for any sequence)
• Probes
• ZEN™ or TAO™ Double-Quenched Probes
• Wide selection of dyes
• Free design tools
• PrimerQuest® Tool
• RealTime Tool
Everything but your sample
2
• Master mix
• PrimeTime® Gene Expression MasterMix
(for probe-based assays)
• Controls
• gBlocks® Gene Fragments
• Water and buffers
• Nuclease-Free Water
• IDTE Buffer

3. Probe-based and primer-only assays
PrimeTime® qPCR Assays
3

4. Use PrimeTime® assays in probe-based and
primer-only experiments
4
• 5′ nuclease, probe-based assays
(primers and probe set)
• Intercalating dye–based assays
(primers only)

5. Advantages of PrimeTime® qPCR Assays
Select from a database when studying gene expression in human, mouse and rat
• Primer and probe sequences provided with each purchase
• Better design delivers sensitive and high-efficiency assays
• Based on up-to-date sequence information (exon boundaries, splice variants)
• Designed to avoid SNPs
• Low price per reaction
• Stop waiting—assays shipped in 2–4 business days
• PrimeTime® qPCR Assays are synthesized when the order is received
• Ensures you have assays designed with the most up-to-date sequence information
• All assay components undergo QC by mass spectrometry; probes are HPLC purified
5

6. Cq values from PrimeTime® assays are equal to or approximately 1 Cq lower than from Competitor A assays
Better design delivers sensitive and efficient assays
0
5
10
15
20
25
30
M
e
a
n
C
q
V
a
lu
e
Gene RefSeq Accession Number
Comparison of Mean Cq Value
PrimeTime
Competitor A
66

7. IDT’s qPCR assay guarantee
Quality guaranteed
7
“PrimeTime®
qPCR
Assays
will
perform
with
an
efficiency
between
90
and
110%
when
tested
over
a
5
log
dynamic
range
with
an
R2 fit
of
≥0.99,
or
the
assay
will
be
replaced
at
no
additional
charge.”

8. PrimeTime® Custom qPCR Assays
Not working with human, mouse, or rat genomes?
Have a unique sequence?
• Use our free PrimerQuest® Tool to design the best possible primer or probe targeting any
sequence of interest
— Easily design:
• With specific primer, probe, or amplicon criteria
• Across a specified location
• Around a fixed primer or probe location
— Software allows customization of ~45 parameters
— Design algorithm includes multiple checks to reduce primer-dimer formation
8
www.idtdna.com/primerquest

9. PrimeTime® Probes
9

10. Choose your dye
10
‡
For
reference
only.
Not
available
through
IDT:
VIC,
NED,
ABY,
PET,
JUN,
LIZ
dyes.
• PrimeTime® qPCR Assays
FAM, HEX™, TET™, Cy® 5
• PrimeTime Custom qPCR Probes
FAM, HEX, TET, Cy 5,
JOE, Yakima Yellow®, Cy 3,
Texas Red®, Tex 615™
• ATTO™ and AlexaFluor® dyes

11. Choose your quencher
11‡
For
reference
only.
Not
available
through
IDT.
• Recommended non-fluorescent quenchers
ZEN™ + Iowa Black® FQ
TAO™ + Iowa Black RQ
Iowa Black RQ

12. Find the appropriate dyes for your instrument
12

13. Use the next generation double-quenched probes
13
Dyes Internalquencher 3′ quencher
ZEN™ Double-QuenchedProbe: FAM,
TET™,
HEX™,
MAX™,
JOE ZEN™ IowaBlack® FQ
TAO™ Double-Quenched Probe: Cy®
5 TAO™ IowaBlack® RQ

14. ZEN™ Quencher: Low background delivers brighter probes
ZEN™ Double-Quenched Probes have lower background and increased sensitivity
(availablewith PrimeTime® qPCR Assays)
14

15. PrimeTime® Gene Expression
Master Mix
2X concentrated master mix for 2-step, probe-based assays
15

16. PrimeTime® Gene Expression Master Mix:
For probe-based assays
• 2-step RT-qPCR
• Direct analysis of DNA
• Singleplex or multiplex assays
• Fast or standard cycling conditions
• Overnight experiments (e.g., high-throughput experiments)
16

17. PrimeTime® Gene Expression Master Mix provides
convenience
• Use with your existing qPCR assays
– Optimized to work with PrimeTime® qPCR Assays from IDT
– Also works with:
• Custom primers/probe sets from IDT
• qPCR assays from other manufacturers
• Use with your existing real-time PCR instrument
– Use with standard or fast cycling conditions
– Use with or without reference dye
• Incorporate
into
kits
– Does
not
require
licensing
fees
17

18. Consistent results from different lots support high
throughput qPCR
18
• Different batches of master mix generate
reproducible results. The standard
deviations of the Cq values for template
levels >10 copies were <0.5.
• Consistent results, even after extended
time at room temperature, means you can
let your PCR experiments run overnight.

19. Part A: Consistent results between singleplex and duplex
reactions—PrimeTime® qPCR Assays with Gene Expression Master Mix
19
Amplification
plot:
•
Singleplex,
black
curves
•
Duplex,
red
curves
Assay
IDs
HPRT:
Hs.PT.58v.45621572
GUSB:
Hs.PT.58v.27737538

20. Part B: Consistent singleplex and duplex reactions–
other commercial assay with the PrimeTime® Gene Expression Master Mix
20
Amplification
plot:
•
Singleplex,
black
curves
•
Duplex,
red
curves

21. Thank you!
www.idtdna.com/PrimeTime
www.idtdna.com/qPCRprobes
www.idtdna.com/qPCRmastermix
www.idtdna.com/DECODED
21

22. Supplemental slides:
— GMP–licensing info
— ZEN™ Double-Quenched Probe applications
— PrimeTime® master mix data
— Instrument dye calibration protocols
— Additional qPCR products for genotyping
22

23. No licensing fees
• PrimeTime® Gene Expression Master Mix
• GMP oligos
– Freedom™ dyes
– Iowa Black® dark quenchers
– ZEN™ and TAO™ quenchers
GMPinfo@idtdna.com
Licensing
fees
and
royalties
increase
as
sales
of
potential
kits
increase—using
license-­‐free
dyes,
quenchers,
and/or
master
mix
could
have
a
positive
effect
on
profitability.
23

24. ZEN™ Double-Quenched Probes: Advantage in
multiplex assays
“The ZEN Double-Quenched Probes have allowed
us to continue adding probes to the already
complex PRRSV assay mix to accommodate the
changing viral landscape. This IDT product has
helped us address the addition of new probes
and primer sets to our assay very quickly.”
—Dr Nelson, Founder of Tetracore
“In addition to providing us with the ZEN™
Double-Quenched Probes to help resolve our
background issues, IDT has been invaluable, both
from a quality and turnaround time perspective.”
—Rolf Rauh, Scientist at Tetracore
24
No bleed over signal with ZEN™
Double-Quenched Probes
Bleed-over signal from
poorly quenched probes

25. “Including the ZEN molecule as a second
quencher has contributed significantly to
increasing our signal-to-noise ratio.
When using dual-quenched probes, the negative
drops emit less fluorescence than typically
observed using standard probes with a single
quenching moiety.
This provides a greater degree of separation
between positive and negative clusters allowing
for more precise counting of droplets in each
population.”
—Coren Milbury
Sr. Scientist, Raindance Technologies
ZEN™ Double-Quenched Probes: For digital PCR
25

26. “Performing MS-HRM or any kind of bisulfite sequencing reaction
results in sequences with reduced cytosine content, and thus
lower Tm.
This makes ZEN probes a great choice for these types of assays
as the Tm of these longer probes can be kept high.
We switched to IDT PrimeTime qPCR Assays because the
PrimeTime qPCR Assays work really well. They are much more
cost efficient, and I really like how we can get them in different
sizes so that when we are testing gene target assays, we can get
just a small scale (Mini Assays), at a lower cost, to see if it would
work for us. That was really appealing to us as a customer.”
Dr. Antoinette Perry,
Trinity College , Dublin
ZEN™ Double-Quenched Probes: Maximum quenching with
long probes for low-complexity sequences
Methylation studies
26

27. Difference in Cq between PrimeTime® Gene Expression
Master Mix and other master mixes
Same threshold and same y-axis
PrimeTime® Gene Expression MM –Alt MM
cDNA
(ng)
∆Cq
gBlocks®
Fragments
(copies)
∆Cq
50 –2.022 107 –2.463
10 –2.169 106 –2.675
2 –2.679 105 –3.098
0.4 –2.386 104 –3.096
0.08 –2.400 103 –3.169
102 –3.784
101 –3.862
cDNA
PrimeTime Gene Expression Master Mix Alternative master mix
gBlocks®Fragments
PrimeTime HPRT Assay ID: s.PT.58v.45621572 27

28. • An overlay of the amplification plots
for the PCRs using unheated or
heated master mix shows no effect
on the amplification curves.
PrimeTime® Gene Expression Master Mix—Exceptional stability
Reproducible amplification after heating the master mix at 55°C
28

29. • Applied Biosystems® 7900 Fast Real-Time
PCR System
• HEX™ Dye
• JOE Dye
• Applied Biosystems® 7300/7500/7500
Fast Real-Time PCR Systems
• HEX™ Dye
• JOE Dye
• TET™ Dye
Download from www.idtdna.com/protocols
Dye calibration protocols
29

30. Additional qPCR products
30
• Genotyping
• LNA PrimeTime® Probes and
Mini LNA PrimeTime® Probes
• rhPrimers & RNase H2
• MGB Eclipse® Probes*
*
Licensed
under
MGB
Eclipse
patents
for
use
by
end-­‐users
in
human
IVD
applications.
For
use
as
a
component
in
the
Purchaser's
IVD
applications
only.
MGB
Eclipse
is
a
registered
trademark
of
Elitech Group.

31. Overview slides:
— PrimeTime® master mix
— PrimeTime qPCR Assays
— PrimerQuest® Tool
— SciTools (free, online software)
— Online resources
— Customer and technical support
— Social media
31

35. Make the most of your time in the lab.
www.idtdna.com/scitools.
• qPCR and PCR amplification
• Gene regulation and knockdown
• MicroRNA Analysis
• Gene design
• Sequencing
• Oligo dilution and resuspension
Our intuitive analysis and design tools
and calculators are available for:
Begin designing at

36. Begin exploring at
Our Knowledge.
YOUR RESOURCE.
www.idtdna.com/support.
Frequently Asked Questions
IDT Publications
Educational Resources
Technical Reports
User Guides & Protocols
Quick References
Video Library
Our online newsletter-DECODED
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37. “Best tech
support ever,
@idtdna!”
Questions?
TALK TO A PERSON.
Lauren SakowskiOur experts are available for consultation.
“The people
at @idtdna are
awesome. A+ for
customer service.”
Nikolai Braun
Contact us by web chat, email, or phone.
Find local contact details at: www.idtdna.com

38. Join the conversation.