High efficiency qPCR with PrimeTime® Gene Expression Master Mix from IDT
-
Upload
integrated-dna-technologies -
Category
Science
-
view
246 -
download
0
Transcript of High efficiency qPCR with PrimeTime® Gene Expression Master Mix from IDT
Dr Erik Wendlandt Scientific Applications Specialist9/28/2016
High efficiency qPCR with PrimeTime® Gene Expression Master Mix
Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
2
3
Primers onlyFor use with intercalating dyes, such as SYBR® Green
Primers and probeFor use in the 5′-nuclease assay
5′-nuclease assays vs. Intercalating-dye
4
Primers onlyFor use with intercalating dyes, such as SYBR® Green
Primers and probeFor use in the 5′-nuclease assay
5′-nuclease assays vs. Intercalating-dye
The PrimeTime® Gene Expression Master Mix has been optimized for use with 5 -nuclease assays′
Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer ®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
5
PrimeTime® Gene Expression Master Mix
Ready-to-use, 2X concentrated master mix, designed for use in 2-step, probe-based RT-qPCR assays.
• Antibody-mediated, hot-start DNA polymerase • dNTPs • MgCl2 • Enhancers and stabilizers
6
• PrimeTime Gene Expression Master Mix has been formulated to give optimal performance with PrimeTime qPCR Assays
• Predesigned PrimeTime qPCR Assays are available for human, mouse, and rat • Custom qPCR design tools are also
available• Use the same master mix for
singleplex/multiplex, fast/standard cycling conditions
• Exceptional benchtop stability • Obtain consistent results from each batch
for high-throughput and overnight runs
Why use the PrimeTime® Gene Expression Master Mix?
7
www.idtdna.com/order/Predesignedassay.aspx
Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
8
The PrimeTime® Gene Expression Master Mix outperforms other commercially available master mixes
9
Comparison of IDT PrimeTime® Gene Expression Master Mix and commercially available master mixqPCR assays consisting of:• PrimeTime Gene Expression Master Mix
with reference dye, or commercially available master mix
• Varying amounts of cDNA (0.016–50 ng) or gBlocks® Gene Fragments (107–101 copies)
The PrimeTime® Gene Expression Master Mix outperforms other commercially available master mixes
10
Comparison of IDT PrimeTime® Gene Expression Master Mix and commercially available master mixqPCR assays consisting of:• PrimeTime ® Gene Expression Master Mix
with reference dye, or commercially available master mix
• Varying amounts of cDNA (0.016–50 ng) or gBlocks® Gene Fragments (107–101 copies)
The PrimeTime® Gene Expression Master Mix consistently outperforms other commercially available master mixes
Successful singleplex and duplex amplification using PrimeTime® Gene Expression Master Mix
11
Singleplex and duplex PCR reactions were performed with: • PrimeTime Gene Expression Master Mix
with reference dye• Assays:
– Predesigned PrimeTime qPCR HPRT AssayAssay ID: Hs.PT.58v.45621572
– Predesigned PrimeTime qPCR GUSB Assay Assay ID: Hs.PT.58v.27737538
• gBlocks® Gene Fragments 107–101 copies
Successful singleplex and duplex amplification using PrimeTime® Gene Expression Master Mix
12
Singleplex and duplex PCR reactions were performed with: • PrimeTime Gene Expression Master Mix
with reference dye• Assays:
– Predesigned PrimeTime qPCR HPRT AssayAssay ID: Hs.PT.58v.45621572
– Predesigned PrimeTime qPCR GUSB Assay Assay ID: Hs.PT.58v.27737538
• gBlocks® Gene Fragments 107–101 copies
Efficient amplification when used with PrimeTime® qPCR assays
PrimeTime® Gene Expression Master Mix also works well with assays purchased from other vendors
Singleplex and duplex PCR reactions were performed with: • PrimeTime Gene Expression Master
Mix with reference dye• Assays:
– Inventoried HPRT and GUSB qPCR assays from supplier AcDNA Template 0.0032–50 ng
13
PrimeTime® Gene Expression Master Mix also works well with assays purchased from other vendors
Singleplex and duplex PCR reactions were performed with: • PrimeTime Gene Expression Master
Mix with reference dye• Assays:
– Inventoried HPRT and GUSB qPCR assays from supplier AcDNA Template 0.0032–50 ng
Efficient amplification when used with commercially available qPCR assays
14
Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
15
Reliable results using different batches of master mix: PCR held at room temperature for 0, 24, 48, and 72 hr before cycling qPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye• Varying amounts of gBlocks® Gene
Fragments107–102 copies
Consistent results for high-throughput qPCR
16
Consistent results for high throughput qPCR
17
Reliable results using different batches of master mix: PCR held at room temperature for 0, 24, 48, and 72 hr before cyclingqPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye,• Varying amounts of gBlocks® Gene
Fragments 107–102 copies
The PrimeTime® Gene Expression Master Mix exhibits consistent and precise results at room temperature for up to 72 hr
Consistent results under thermal stress
18
Reliable results using different batches of master mix: 0, 1, 3, 5, and 7 days at 50ºC. qPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye• Varying amounts of gBlocks® Gene
Fragments 107–102 copies
Consistent results under thermal stress
19
Consistent results under thermal stress
20
The PrimeTime® Gene Expression Master Mix exhibits consistent performance after extended heat-stress
PrimeTime® Gene Expression Master Mix is stable after international shipment under ambient conditions
21Data courtesy of Ticiane H Santa Rita and Dr Gustavo B Barra, Laboratório Sabin de Análises Clínicas, Brasília, DF
PrimeTime® Gene Expression Master Mix is stable after international shipment under ambient conditions
22
Shipping conditions do not affect performance of the PrimeTime® Gene Expression Master Mix
Data courtesy of Ticiane H Santa Rita and Dr Gustavo B Barra, Laboratório Sabin de Análises Clínicas, Brasília, DF
Consistent results upon multiple freeze-thaw cycle
23
Reliable results using different batches of master mix after 0, 5, 10, and 20 freeze-thaw cyclesqPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye• Varying amounts of gBlocks® Gene
Fragments 107–102 copies
Consistent results upon multiple freeze-thaw cycle
24
Reliable results using different batches of master mix after 0, 5, 10, and 20 freeze-thaw cyclesqPCR assays consisting of:• PrimeTime® Gene Expression Master Mix,
reference dye,• Varying amounts of gBlocks® Gene
Fragments 107–102 copies
Multiple freeze-thaw cycles do not affect the functionality of the master mix
Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
25
Considerations for multiplexing
26
• Advantages• Save reagent/master mix costs• Analyze a large number of targets in a single reaction• Maximize usage of rare samples
• Considerations• Not all master mixes are formulated with enough polymerase and dNTPs to support
multiplexing—the PrimeTime® Gene Expression Master Mix is compatible with multiplexing• Need high efficiency assay designs for multiplexing
• Validate and optimize individual assays prior to multiplexing• Primers and probes must be assessed for interactions• Carefully select dyes for each assay
• Match the lowest copy target with the brightest fluorophore (FAM)• Lowered plateau in a multiplex vs. singleplex is normal
• Cq values should remain consistent between singleplex vs. multiplex
Multiplexing with the PrimeTime® Gene Expression Master Mix(4-Plex)
Multiplex setupqPCR assays consisting of:• PrimeTime® Gene
Expression Master Mix, reference dye
• Varying amounts of cDNA 0.016–50 ng
28
Multiplexing with the PrimeTime® Gene Expression Master Mix(Overlay)
FAM
Singleplex
Yakima Yellow Cy5 Cy5.5
Singleplex
28
Multiplexing with the PrimeTime® Gene Expression Master Mix(Overlay)
FAM
Singleplex
Yakima Yellow Cy5 Cy5.5
Singleplex
Efficient amplification under singleplex and multiplex conditions
29
Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
30
Primer-dimer analysis with the Oligo Analyzer® Tool
c
31
c
Primer-dimer analysis with the Oligo Analyzer® Tool
32
Primer-dimer analysis with the Oligo Analyzer® Tool
33
Primer-dimer analysis with the Oligo Analyzer® Tool
34
Primer-dimer analysis with the Oligo Analyzer® Tool
35
Primer-dimer analysis with the Oligo Analyzer® Tool
36
Primer-dimer analysis with the Oligo Analyzer® Tool
37
Primer-dimer analysis with the Oligo Analyzer® Tool
38
PrimeTime® Multiplex Dye Selection Tool
39
PrimeTime® Multiplex Dye Selection Tool
40
PrimeTime® Multiplex Dye Selection Tool
41
PrimeTime® Multiplex Dye Selection Tool
42
PrimeTime® Multiplex Dye Selection Tool
43
PrimeTime® Multiplex Dye Selection Tool
44
PrimeTime® Multiplex Dye Selection Tool
45
Outline• Review of 5′-nuclease assays• PrimeTime® Gene Expression Master Mix components• Performance of the PrimeTime Gene Expression Master Mix
• With IDT assays• With commercially available assays
• PrimeTime Gene Expression Master Mix stability• Singleplex vs. multiplexing applications
• Considerations for multiplexing• Useful tools for multiplexing
• Oligo Analyzer®, Multiplex Dye selection Tool• Standard vs. fast cycling conditions
46
Efficiency of qPCR assays under fast and standard cycling conditions using PrimeTime® Gene Expression Master Mix
47
Standard or fast cycling conditionsqPCR assays consisting of:• PrimeTime Gene Expression
Master Mix, reference dye• Varying amounts of cDNA 0.016–
50 ng
Efficiency of qPCR assays under fast and standard cycling conditions using PrimeTime® Gene Expression Master Mix
48
Standard or fast cycling conditionsqPCR assays consisting of:• PrimeTime Gene Expression
Master Mix, reference dye,• Varying amounts of cDNA 0.016–
50 ng
Efficiency of qPCR assays under fast and standard cycling conditions using PrimeTime® Gene Expression Master Mix
49
Standard or fast cycling conditionsqPCR assays consisting of:• PrimeTime Gene Expression
Master Mix, reference dye• Varying amounts of cDNA 0.016–
50 ng
Efficient amplification under standard and fast cycling conditions
Conclusions about the PrimeTime® Gene Expression Master Mix
• Works well for assays from IDT and other suppliers• Efficient amplification independent of the assay source
• Exhibits exceptional temperature and freeze-thaw stability• Reduced packaging resources and shipping costs• Ensured performance for high-throughput and overnight runs
• High efficiency when used for singleplex or multiplex reactions• Maximize use of rare samples and reduce reagent costs
• Performs well under standard or fast cycling conditions• Improved time from set-up to data
50
“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.comOr email: [email protected]
THANK YOU!We will email you the webinar recording and slides next week.