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Table of contents
Introduction ............................................................................................... 3-7 SuperScript III First-Strand Synthesis System for qRT-PCR ........7 qPCR SuperMixes ....................................................................................8-10 Standard ..................................................................................................8-9 SNP genotyping ...................................................................................10 One-Step qRT-PCR Kits ....................................................................... 11-15 One-Step qRT-PCR Kits .................................................................. 11-12 ThermoScriptqRT-PCR Kits .............................................................13 RNA UltraSense qRT-PCR System ..........................................14-15 Two-Step qRT-PCR Kits .......................................................................16-17 qRT-PCR Directly from Cells ..............................................................18-19 For qPCR ........................................................................................... 20-21 For qRT-PCR ............................................................................................ 22 Detection systems ............................................................................. 23-28 Custom D-LUX Fluorogenic Primers................................... 23-26 Certied LUX Fluorogenic Primer Sets ............................. 27-28 qPCR Accessories .................................................................. 19, 26, 29-30 CellsDirect Lysis and Resuspension Buer ................................19 qPCR Instrument Calibration Kit ..................................................... 26 qPCR Plasmid Standards .................................................................... 29 Uracil DNA Glycosylase (UDG) ......................................................... 30 Related Products ........................................................................................ 30
RNA Purication ChargeSwitch Total RNA Cell Kit PureLink Micro-to-Midi Total RNA Purication Systems TRIzol Reagents
RNA Sample QC Quant-iT RNA Assay Kit RiboGreen RNA Assay Kits
Microarray Labeling and Detection SuperScript cDNA Direct and Indirect Labeling Systems SuperScript RNA Amplication Systems RNAi Knockdown Experiments Validated Stealth RNAi DuoPaks Stealth Select 3 RNAi Sets Custom BLOCK-iT siRNA BLOCK-iT RNAi Vectors
qRT-PCR Validation LUX Fluorogenic Primers qPCR SuperMixes One-Step qRT-PCR Kits Two-Step qRT-PCR Kits qPCR Plasmid Standards
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One-step qRT-PCR
One-step qRT-PCR allows easier processing of large numbers of samples from few genes of interest. Both cDNA synthesis and PCR are performed in a single tube using gene-specic primers and RNA target(s) from either RNA or mRNA. All components necessary for qRT-PCR are mixed in the tube, and reverse transcription is directly followed by PCR without additional handling. Reaction tubes are not opened between cDNA synthesis and amplication so carryover contamination is minimized. Furthermore, by amplifying the entire cDNA sample, one-step qRT-PCR enables highly sensitive detection from as few as 10 copies of RNA template, with a broad dynamic range that supports accurate quantication of high-copy mRNA up to 1 g of total RNA. Table 1 compares the benets of one-step and two-step qRT-PCR.
Two-step procedure Prime rst-strand DNA with: Provides: Oligo(dT) primer Random hexamers Flexibility Choice of primers for cDNA synthesis Ability to save cDNA for later use Ability to optimize for dicult qRT-PCR Recommended uses: Ideal for detecting or quantifying multiple genes of interest and/or preservation of cDNA
Convenience Amplication enzymes premixed with reverse transcriptase Fewer pipetting steps reduce chances for contamination Well suited for high-throughput applications Ideal for analyzing large numbers of samples from few genes of interest
DNA, cDNA
qPCR SuperMix, Room Temperature Stable, pages 20-21 qPCR SuperMix, Genotyping, page 10 Two-Step Kits, Standard, pages 16-17 Two-Step Kits, Directly from cells, pages 18-19 One-Step Kits, Standard, pages 11-12 One-Step Kits, Low Abundance Targets, pages 14-15 One-step Kits, Room Temperature Stable, page 22
N/A SuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit w/ROX SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit with SYBR Green SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit w/ROX
SuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit with SYBR Green SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit
RNA
RNA UltraSense One-Step qRT-PCR System SuperScript III Platinum RTS One-Step qRT-PCR Kit w/ROX
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0 5 10 15
20
25
30
Activity assays were run at 37C for 5 hours with Taq DNA polymerase and Platinum Taq DNA Polymerase.
Enzymes were incubated at 94C in the reaction buer provided. Aliquots were removed at the various times and utilized in a standard activity assay.
1X HML
0.01
0.01
10
10
10
10
10
10
10
1000
1000
1000
Dynein (12.3 kb) fibrillin (9.4 kb) adenomatous polyposis (8.5 kb) DNA polymerase (6.8 kb) nuclear receptor coactivator (6.1 kb) guanine nuc. exchange factor (5.9 kb) vinculin (4.6 kb) transcription factor (3.6 kb) B-factor properdin (2.4 kb) GAPDH (532 bp) -actin (353 bp)
cDNA was synthesized from HeLa total RNA or rat total RNA for Dynein with oligo(dT) primer using 400 U of SuperScript III RT at 50C. 10% of cDNA reaction was added to 50-l PCR reaction containing primers for each gene and 2 U of Platinum Taq DNA Polymerase or 1 U of Platinum Taq DNA Polymerase High Fidelity, 35 or 40 cycles, 1 min/kb.
SuperScript II
TM
M-MLV 37 42 45 50 55 M
37 42 45 50 55 37 42 45 50 55
An autoradiograph is shown of 32P-labeled cDNA synthesized from a mixture of 0.25 g of RNA each of 1.35 kb, 2.4 kb, 4.4 kb, 7.5 kb, and 9.5 kb with 200 units of each RT at various temperatures. Lane M is 32P-labeled 1 kb DNA ladder.
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B. Standard Curve
Panel A: 10 to 107 copies of -actin plasmid DNA were amplied with 200 nM each primer and detected with 100 nM TaqMan Probe using Platinum qPCR SuperMix-UDG (red plot) or ABI Universal PCR MasterMix (blue plot). Reactions were assembled at room temperature. PCR incubations were 95C for 10 min., followed by 50 cycles of 95C for 15 sec. and 60C for 1 min. using an ABI PRISM 7700. Panel B: Standard curve showing the starting template amount versus Ct value.
Product For LUX Primers or probe-based detection Platinum qPCR SuperMix-UDG (for any instrument) Platinum qPCR SuperMix-UDG with ROX (for ABI instruments - 7000, 7300, 7700, 7900)
Rxn size 50 l 50 l 50 l 50 l
B. Standard Curve
qPCR of 10-fold serial dilutions (107 to 10 copies) of pCR2.1 plasmid was performed using primers specic to the Kanamycin resistance gene (200 nM each) with Platinum SYBR Green qPCR SuperMix-UDG and ROX Reference Dye. Reactions were incubated for min. at 50C, then 2 min. at 95C, followed by 50 cycles of 95C for 15 sec.; 60C, 2 30 sec. using the ABI PRISM 7700.
Figure 7 Invitrogens
Platinum SYBR Green qPCR SuperMix-UDG with ROX outperforms ABIs Master Mix
7.9 6.9
Invitrogen ABI
Relative uorescence
5.9
4.9 3.9 2.9 1.9 0.9 -0.1
Amplification plots comparing Invitrogens Platinum SYBR Green qPCR SuperMix-UDG w/ROX and ABIs SYBR Green qPCR MasterMix. Invitrogens qPCR Plasmid Standard for High Abundance Genes was used as template at concentrations ranging from 107 to 102 copies were used for detection. Reactions were run on an ABI PRISM 7700 using ABIs recommended cycling protocol. Even with ABIs protocol, the Invitrogen mix outperformed the ABI mix by over 6 Cts.
5 7
9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
Cycle Number
Product For SYBR Green-based detection Platinum SYBR Green qPCR SuperMix-UDG (for any instrument) Platinum SYBR Green qPCR SuperMix-UDG with ROX (for ABI instruments - 7000, 7300, 7700, 7900)
Rxn size 50 l 50 l 50 l 50 l
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Figure 8 Platinum qPCR SuperMix-UDG for SNP Genotyping vs. ABI Universal PCR MasterMix
A. Invitrogen
FAM GG GC
VIC CC GG GC
GC
CC
GG
CC
B. ABI
FAM
GG GC
VIC GG GC GC CC GG
CC
CC
Panel A. qPCR genotyping results using TaqMan SNP Genotyping Assay for BAT 1 and Invitrogens Platinum qPCR SuperMix-UDG for SNP Genotyping. Panel B. qPCR genotyping results using TaqMan SNP Genotyping Assay for BAT 1 and ABIs Universal Master Mix. Each assay was run according to the reagent manufacturers recommended protocol on an ABI PRISM 7900HT.
Product Platinum qPCR SuperMix-UDG for SNP Genotyping (for any instrument)
Rxn size 20 l 20 l
10
Invitrogen
ABI
y = -3.45(x) + 44.288
R 2 = 0.9914
Invitrogen ABI
B.
40
Invitrogen ABI 35
Ct Values
30
25
0.1 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
15 1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
Cycle Number
Panel A: Amplication plots comparing Invitrogens SuperScript III Platinum One-Step qRT-PCR SuperMix with ROX and ABIs TaqMan One-Step RT-PCR MasterMix. HeLa total RNA in ten-fold serial dilutions from 100 ng to 1 pg was used as template, and reactions were run on an ABI PRISM 7700 using ABIs recommended cycling protocol. Even with ABIs protocol, the Invitrogen reagents outperformed the ABI reagents by over 4 Cts. Panel B: Standard curves generated from Ct values vs. relative template copy numbers of HeLa total RNA. Copy numbers are based on ten-fold serial dilutions of HeLa total RNA template. Amplications were done using -actin TaqMan probes on an ABI PRISM 7700 Instrument following ABIs recommended protocol. Even with ABIs protocol, the Invitrogen reagents outperformed the ABI reagents (.9993 vs. .9914).
Figure 10 Get greater sensitivity with the SuperScript III Platinum One-Step qRT-PCR Kit
A. Amplication Plot
SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) Quantitect Probe RT-PCR Kit (Qiagen)
40
B. Standard Curve
y = -3.286x + 29.173 R2 = 0.9968 y = -3.553x + 31.708 R2 = 0.9966
35
30
Rn
25
20
15
Cycle Number
The human -actin target was quantified by qRT-PCR using 10-fold serial dilutions of total HeLa RNA (100 ng to 0.1 pg) with either the SuperScript III Platinum One-Step qRT-PCR Kit from Invitrogen (red) or the Quantitect Probe RT-PCR Kit from Qiagen (blue), 200 nM each amplification primer, 100 nM TaqMan Probe, and ROX Reference Dye. cDNA synthesis was performed at 50C for 15 min, followed by 40 cycles of PCR using an ABI PRISM 7700.
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Cycle Number
B. Standard Curve
40 35 30
10 4 pg
10 3 pg 10 2 pg 10 pg 1 pg 0.1 pg
25 20 15 10 5
Rn
0.1
0.01 0 5 10 15 20 25 30 35 40 45 50
Cycle Number
qPCR of 10-fold serial dilutions (105 to 0.1 pg) of total HeLa RNA with 200 nM each GAPDH primer using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit. All reactions were performed on the ABI PRISM 7000 using the following protocol: 3 min RT at 50C, followed by 50 cycles of 95C for 15 sec and 60C for 30 sec.
Figure 12 The SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit outperforms the competition
A. Amplication Plot vs QuantiTect
100
_ SuperScript III Platinum SYBR Green One-Step qRT-PCR (Invitrogen) _ QuantiTect SYBR Green RT-PCR (Qiagen)
10
10
Rn
Rn
1 0.1 0 5 10 15 20 25 30 35 40 45 50 0.1 0 5 10 15 20 25 30 35 40 45 50
Cycle Number
Cycle Number
Panel A: qPCR of 10-fold serial dilutions (10 ng to 1 pg) of total HeLa RNA was performed with 200 nM each -Actin primer using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit from Invitrogen (red lines; standard curve: y = -3.57x + 31.05; R2 = 0.998) or the QuantiTect SYBR Green RT-PCR Kit from Qiagen (blue lines; standard curve: y = -3.612x + 32.10; R2 = 0.981) according to each manufacturers protocol. Reactions were performed on the ABI PRISM 7000. Panel B: qPCR of 10-fold serial dilutions (10 ng to 1 pg) of total HeLa RNA was performed with 200 nM each GAPDH primer using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit from Invitrogen (red lines; standard curve: y = -3.52x + 31.15; R2 = 0.995) or the Brilliant SYBR Green qRT-PCR Kit from Stratagene (blue lines; standard curve: y = -3.893x + 32.89; R2 = 0.996) according to each manufacturers protocol. Reactions were performed on the ABI PRISM 7000.
Product For LUX primers or probe-based detection SuperScript III Platinum One-Step qRT-PCR Kit (for any instrument) SuperScript III Platinum One-Step qRT-PCR Kit with ROX (for ABI instruments - 7000, 7300, 7700, 7900) For SYBR Green-based detection SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (for any instrument) SuperScript III Platinum with SYBR Green One-Step qRT-PCR Kit with ROX (for ABI instruments - 7000, 7300, 7700, 7900)
Quantity 100 rxns 500 rxns 100 rxns 500 rxns 100 rxns 500 rxns 100 rxns 500 rxns
Rxn size 50 l 50 l 50 l 50 l 50 l 50 l 50 l 50 l
Cat. no. 11732-020 11732-088 11745-100 11745-500 11736-051 11736-059 11746-100 11746-500
12
ThermoScript RT
512 bp 414 bp 344 bp 225 bp
AMV RT
512 bp 414 bp 344 bp 225 bp
cDNA was synthesized from 10 ng total RNA using 15 units of ThermoScript RT or 15 units AMV RT according to manufacturers recommendations. One tenth of each reaction was amplified with Platinum Taq DNA Polymerase High Fidelity.
B. Standard Curve
qRT-PCR of a 145-bp -actin fragment was performed with the Platinum qRT-PCR ThermoScript One-Step System (blue) or Supplier P Quantitative One-Step RT-PCR System (red) according to manufacturers recommendations. Starting RNA varied from 5 g to 50 fg in 10-fold serial dilutions. Normalized relative fluorescence (Rn) was collected using TAMRA as the passive reference. Panel A: Linear scale amplification plots. Panel B: Standard curve plots.
Rxn size 50 l 50 l
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Figure 15 High-sensitivity RNA detection and qRT-PCR eciency with the RNA UltraSense One-Step qRT-PCR System
Amplication of mChAT from Brain RNA
1.00E+00
25 ng/l brain RNA
Total RNA from mouse brain was diluted to 25 ng/l, 2.5 ng/l, 0.25 ng/l and 25 pg/l. Thirteen microliters of each dilution were added to triplicate 20-l reactions. The RNA UltraSense One-Step qRT-PCR System was able to amplify the rare ChAT message from RNA samples as dilute as 25 pg/l with a PCR efficiency of 97%.
Rn
Cycle #
14
Figure 16 The RNA UltraSense qRT-PCR System provides improved amplication of viral RNA with signicant secondary structure
A
3.50E-01
1e5 copies viral RNA 1e4 copies viral RNA
1e3 copies viral RNA 1e2 copies viral RNA
No Template Control
B
3.75E+00
3.00E-01
3.25E+00
2.50E-01
2.75E+00
2.00E-01
2.25E+00
Rn
Rn
1.50E-01
1.75E+00
1.00E-01
1.25E+00
5.00E-02
7.50E-01
0.00E+00
2.50E-01
-5.00E-02 0 5 10 15 20 25 30 35 40 45 50
-2.50E-01 0 5 10 15 20 25 30 35 40 45 50
Cycle #
Cycle #
The Newcastle virus is a single-stranded RNA virus that exhibits considerable secondary structure. qRT-PCR was performed on 105, 104, 103, 102, or 101 copies of END RNA using TaqMan primers and probe against viral sequence. Immediately before loading, the templates were heated to 95C for 5 minutes and then placed on ice. Panel A: RNA UltraSense One-Step qRT-PCR System with SuperScript III RT provides clean amplification of four serial dilutions of RNA with exceptional efficiency (107%). A standard, competing one-step qRT-PCR system exhibits poor performance when encountering significant secondary structure. The standard system is only able to amplify two serial dilutions of RNA template. Panel B: Amplification of 6 serial dilutions of pLenti6/V5-GFP viral RNA with 90% efficiency.
Rxn size 50 l
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Figure 17 The SuperScript III Platinum Two-Step qRT-PCR Kit provides specic detection with LUX Fluorogenic Primers
A. Amplication Plot
HeLa cDNA 100 ng 10 ng 1 ng 100 pg 10 pg
40 35
B. Standard Curve
Rn
0
10
15
20
25
30
35
40
45
50
Cycle number
The human SDHA gene was quantied by qPCR using 10-fold serial dilutions (100 ng to 10 pg) of HeLa cDNA with 200 nM JOE-labeled LUX Primer, 200 nM unlabeled LUX Primer, SuperScript III Platinum Two-Step qRT-PCR Kit, and ROX Reference Dye. Reactions were incubated for 2 min. at 50C, then 2 min. at 95C followed by 50 cycles of 95C for 15 sec.; 60C for 30 sec. using an ABI PRISM 7700.
Figure 18 The SuperScript III Platinum Two-Step qRT-PCR Kit provides sensitive detection with TaqMan Probes
A. Amplication Plot
HeLa cDNA
30 25 20 15 10 5 0
B. Standard Curve
50 ng 5 ng 500 pg 50 pg 5 pg
Rn
10
20
30
40
50
50
5 x 102
5 x 103
5 x 104
Cycle Number
Human 18S rRNA was quantied by qPCR using 10-fold serial dilutions (50 ng to 5 pg) of HeLa cDNA with 200 nM each amplication primer, 100 nM TaqMan Probe, SuperScript III Platinum Two-Step qRT-PCR Kit, and ROX Reference Dye. Reactions were incubated for 2 min. at 50C, then 2 min. at 95C followed by 50 cycles of 95C for 15 sec.; 60C for 30 sec. using an ABI PRISM 7700.
16
30 25
B. Standard Curve
100 ng 10 ng 1 ng 100 pg 10 pg
20 15 10 5 0
Rn
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35
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45
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10
102
103
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105
Cycle number
The human GAPDH gene was quantied by qPCR using 10-fold serial dilutions (100 ng to 10 pg) of HeLa cDNA with 200 nM each amplication primer, SuperScript III Platinum Two-Step qRT-PCR Kit with SYBR Green, and ROX Reference Dye. Reactions were incubated for 2 min. at 50C, then 2 min. at 95C followed by 50 cycles of 95C for 15 sec.; 60C for 30 sec. using an ABI PRISM 7700.
Product For LUX Primers or probe-based detection SuperScript III Platinum Two-Step qRT-PCR Kit (for any instrument) SuperScript III Platinum Two-Step qRT-PCR Kit with ROX (for ABI instruments - 7000, 7300, 7700, 7900) For SYBR Green-based detection SuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit (for any instrument) SuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit with ROX (for ABI instruments - 7000, 7300, 7700, 7900)
Quantity 100 rxns 500 rxns 100 rxns 500 rxns 100 rxns 500 rxns 100 rxns 500 rxns
Rxn size 50 l 50 l 50 l 50 l 50 l 50 l 50 l 50 l
Cat. no. 11734-050 11734-068 11747-100 11747-500 11735-032 11735-040 11748-100 11748-500
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Figure 20 Single-cell detection with the SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit
A. Amplication Plot using GAPDH with 1 cell
1
Template Quantity _ 2 l cDNA _ 4 l cDNA _ 6 l cDNA _ 8 l cDNA
10
Rn
0.1
Rn
0.1 0.01
0.01
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Cycle Number
Cycle Number
qPCR was performed on 2, 4, 6 (GAPDH only), and 8 l of cDNA synthesized from a single HeLa cell (by dilution) using the SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit with 200 nM each primer and 100 nM TaqMan Probe specific to either GAPDH (Panel A) or TFRC (Panel B) housekeeping genes. Fifty-microliter reactions were incubated for 2 min. at 50C, then 2 min. at 95C, followed by 50 cycles of 95C for 15 sec.; 60C for 30 sec. using the ABI PRISM 7700.
Figure 21 SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit detects up to 10,000 cells
A. Amplification Plot using GAPDH with 1 0,000 cells
1
Rn
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0.01 10 15 20 25 30 35 40 45 50
Rn
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Cycle Number
Cycle Number
qPCR was performed on 10-fold serial dilutions (undiluted to 1:10,000) of cDNA synthesized from 10,000 HeLa cells using the SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit. Four microliters of each dilution were added to a 50-l qPCR reaction containing 200 nM each primer and 100 nM TaqMan Probe to GAPDH (Panel A; standard curve: y = -3.37x + 36.315; R2 = 0.9967) or TFRC (Panel B; standard curve: y = -3.395(x) + 41.5; R2 = 0.999) housekeeping genes. Reactions were incubated for 2 min. at 50C, then 2 min. at 95C followed by 50 cycles of 95C for 15 sec.; 60C for 30 sec. using the ABI PRISM 7700.
18
Cycle Number
Rn
Rn
0.1
0.1
0.01 15 20 25 30
35
0.01
40 45 50
15
20
25
30
35
40
45
50
Cycle Number
Cycle Number
qPCR was performed on 10-fold serial dilutions of cDNA synthesized from HeLa cells using the SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit from Invitrogen (red) or the Cells-to-cDNA II Kit from Ambion (blue). Four microliters of each dilution were added to a 50-l qPCR reaction containing 200 nM each TaqMan primer and 100 nM probe specific to GAPDH and amplified with a standard protocol on the ABI PRISM 7700. Panel A: Results comparing cDNA synthesized from 1,000 cells with the SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit (standard curve: y = 3.48x + 39.376; R2 = 0.9968) or 10,000 cells with the Cells-to-cDNA II Kit (standard curve: y = -3.33x + 39.047: R2 = 0.9982). Because of protocol differences, the Cells-to-cDNA II Kit requires 10X more starting sample to produce the cDNA equivalent of 1,000 cells. Panel B: Results Cycle Number comparing cDNA synthesized from 1,000 cells with the SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit (standard curve: y = -3.48x + 39.376; R2 = 0.9968) and 1,000 cells with the Cells-to-cDNA II Kit (standard curve: y = -3.35x + 43.367: R2 = 0.995) following each manufacturers suggested protocol.
Figure 23The SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit provides sensitive detection with LUX Primers or SYBR Green I
A. Amplication plot using LUX Primers
1
Rn
Rn
0.1
0.01 15 20 25 30 35 40 45 50
0.1 0 5 10 15 20 25 30 35 40 45 50
Cycle Number
Cycle Number
qPCR was performed on 10-fold serial dilutions of cDNA synthesized from 1000 HeLa cells using the SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit. Four microliters of each dilution were added to a 50-l qPCR reaction with ROX Reference Dye and the reagents indicated. Reactions were incubated for 2 min. at 50C, then 2 min. at 95C, followed by 50 cycles of 95C for 15 sec.; 60C for 30 sec. using the ABI PRISM 7700. Panel A: Amplification and detection using 200 nM each LUX Primer specific to the SDHA housekeeping gene. Standard curve: y = -3.539x + 42.808; R2 = 0.99 Panel B: Amplification and detection with SYBR Green using 200 nM each primer specific to the -actin housekeeping gene. Standard curve: y = -3.375x + 29.348; R2 = 0.995
Product For LUX Primers-based and probe-based detection SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit (for any instrument) For SYBR Green-based detection SuperScript III Platinum CellsDirect Two-Step qRT-PCR Kit with SYBR Green (for any instrument) CellsDirect Resuspension and Lysis Buer (includes 10 ml resuspension buer and 1 ml lysis buer)
Quantity 100 rxns 500 rxns 100 rxns 500 rxns 1 Kit
Rxn size 50 l 50 l 50 l 50 l
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Figure 24 RTS Amplication Reagents speed reaction setup Standard Liquid Reagents Gather reagents and ice Thaw buffers Add primers and template on ice Begin incubation Elapsed time: 22 min. Begin incubation Elapsed time: 8 min. RTS Reagents Add primers and template Resuspend for 1 minute
Figure 25 Platinum RTS qPCR SuperMix-UDG (blue) provides similar performance to liquid Platinum qPCR SuperMix-UDG (red) with LUX Fluorogenic Primers and TaqMan Probes
A. Amplication plot using LUX Primers (Melting Curve inserted)
Y= -3.45x + 38.7; R2 = 0.998 Y= -3.39x + 38.5; R2 = 0.997
B. Amplication Plot using TaqMan Probes
84C
Rn
10
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25 Cycle Number
30
35
40
Rn
10
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25 Cycle Number
30
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qPCR of 10-fold serial dilutions (100 ng-10 pg) of HeLa cDNA was performed using 200 nM each LUX Primers (Panel A) or 200 nM each primer and 100 nM TaqMan Probe (Panel B) specific to the -actin housekeeping gene with Platinum qPCR SuperMix-UDG (red) or with Platinum RTS qPCR SuperMix-UDG (blue) and ROX Reference Dye. Reactions were incubated for 2 min. at 50C, then 2 min. at 95C, followed by 40 cycles of 95C for 15 sec.; 60C for 30 sec. using the ABI PRISM 7700.
20
82
84
82
84
Rn
D Rn 10
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30
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40
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20
25 Cycle Number
30
35
40
Panel A: qPCR of 10-fold serial dilutions (100 ng-10 pg) of HeLa cDNA was performed using primers (200 nM each) specific to the GAPDH housekeeping gene with Platinum SYBR Green qPCR SuperMix-UDG and additional BSA (red) or with Platinum RTS SYBR Green qPCR SuperMix-UDG (blue). Reactions were incubated for 1 min. at 50C, then 1 min. at 92C, followed by 40 cycles of 95C for 5 sec.; 60C for 30 sec. using the Roche LightCycler. Panel B: qPCR of 10-fold serial dilutions (100 ng-10 pg) of HeLa cDNA was performed using primers (200 nM each) specific to the GAPDH housekeeping gene with Platinum SYBR Green qPCR SuperMix-UDG (red) and or with Platinum RTS SYBR Green qPCR SuperMix-UDG (blue) and ROX Reference Dye. Reactions were incubated for 2 min. at 50C, then 2 min. at 95C, followed by 40 cycles of 95C for 15 sec.; 60C for 30 sec. using the ABI PRISM 7000.
Product For LUX Primers or probe-based detection Platinum RTS qPCR SuperMix-UDG (for any instrument) Platinum RTS qPCR SuperMix-UDG with ROX (for ABI instruments - 7000, 7300, 7700, 7900) For SYBR Green-based detection Platinum RTS SYBR Green qPCR SuperMix-UDG (for any instrument) Platinum RTS SYBR Green qPCR SuperMix-UDG 96 with ROX (for ABI instruments - 7000, 7300, 7700, 7900)
Rxn size 25 l 25 l 25 l
25 l 25 l 25 l
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Rn
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Rn
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Cycle Number
Cycle Number
qPCR of 10-fold serial dilutions (106 to 1 pg) of total HeLa RNA was performed with TaqMan primers and probe for GAPDH labeled with JOE-BHQ using the SuperScript III Platinum RTS One-Step qRT-PCR Kit with lyophilized reagents (A) or the standard liquid SuperScript III Platinum One-Step qRT-PCR Kit (B). All reactions were performed on the ABI PRISM 7000.
Figure 28 SuperScript III Platinum RTS One-Step qRT-PCR Kit provides sensitive amplication on the Roche LightCycler
A. Amplication Plot
Total HeLA RNA
_
B. Standard Curve
40 35
Cycle Threshold
Rn
30 25 20 15 10 1 10
10
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40
102
103
104
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106
Cycle Number
qPCR of 10-fold serial dilutions (106 to 1 pg) of total HeLa RNA was performed with TaqMan primers and probe for -Actin labeled with FAM-TAMRA using the SuperScript III Platinum RTS One-Step qRT-PCR Kit on the Roche LightCycler.
Product SuperScript III Platinum RTS One-Step qRT-PCR Kit (for any instrument) SuperScript III Platinum RTS One-Step qRT-PCR Kit with ROX (for ABI instruments - 7000, 7300, 7700, 7900)
Rxn size 25 l 25 l 25 l
22
B
Threshold Cycle (Ct)
Rn
0.1
0.01 1 6 11 16 21 26 31 36
Rn
0.1 0.01 1 6 11 16 21 26 31 36
Cycles
Cycles
-dF/dT
0.05 0.04 0.03 0.02 0.01 0 45.7 51.1 56.2 61.3 66.4 71.5
0
76.7
81.8
86.9
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Temperature ( C)
Side-by-side comparison of qPCR with LUX Primers designed using D-LUX Designer (A) and TaqMan probe detection system using ABIs Gene Expression Assay (B) for human Human NROB2 (NM_021969) . Amplication was performed using Platinum qPCR SuperMix-UDG using primer and probe amounts recommended by manufacturers. Human HMBS ORF clone was used with serial 10-fold dilution starting at 10 7 copies per reaction. Reactions were incubated for 2 min at 50C, 2 min at 95C, and 50 cycles of 15 sec 95C, 30 sec at 55C, and 30 sec at 72C, followed by a melting curve using an ABI PRISM 7700 instrument.
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Figure 31Melting curve analysis conrming specic amplication with LUX Primers
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qPCR was performed on 10-fold serial dilutions (106 to 103 copies) of human -actin plasmid using 200 nM FAM-labeled LUX Primer, 200 nM unlabeled primer, and Platinum qPCR SuperMix-UDG. Reactions were amplified by 50 cycles of PCR using a Corbett Research Rotor-Gene. Melting curve analysis was performed after amplification using a temperature ramp of 1C/5 sec. between 65C and 95C. Colored lines indicate template-specific reactions. Black lines indicate no-template controls.
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Figure 32Multiplexing is simple and ecient with LUX Primers A. (CtAmplication Plot A. ) Amplication Plot B. Standard Curve B. Standard Curve
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y =-3.424(x) + 39.752 R 2 = 0.991
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Multiplex qPCR with LUX Primers was performed using Platinum qPCR SuperMix-UDG supplemented to a final concentration of 6 mM MgCl2. 200 nM FAM-labeled LUX SDHA Primers were used for amplification of serial dilution of human cDNA and 100 nM JOE-labeled LUX GAPDH Primers were used for amplification of GAPDH added to the multiplex assays at a constant amount of 105 copies per assay. Reactions were incubated for 2 min at 50C, 2 min at 95C and 50 cycles of 15 sec 95C, 30 sec 55C, 30 sec 72C using an ABI PRISM 7700.
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Quantity 1 kit
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Certied LUX Primer Sets for Housekeeping Genes Accurate real-time normalization
Certified LUX Primer Sets for Housekeeping Genes are designed for use in conjunction with custom-designed, target-specific LUX Fluorogenic Primers to provide reliable normalization for qPCR. Each premade primer set has been functionally validated and optimized for accurate quantification of a corresponding stable control gene with reliable normalization data. The wide range of genes, species, and expression levels represented enables you to match the expression characteristics of your target gene, for greater normalization accuracy. Certified LUX Primer Sets for Housekeeping Genes: Offer sensitive, specific, cost-effective normalization in a ready-to-use format Are functionally validated for human, mouse/rat, and Drosophila control genes Represent multiple expression levels to match your target expression level Are available with either FAM or JOE labels For a complete list of available primer sets please visit us at www.invitrogen.com/lux or use our database search at www.invitrogen.com/certifiedluxsearch.
Product Certied LUX Primer Sets for Human Genes, FAM labeled Certied LUX Primer Sets for Housekeeping Genes, FAM labeled Certied LUX Primer Sets for Housekeeping Genes, JOE-labeled
Rxn size 50 l 50 l 50 l
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0.1
Serial dilution of cDNA from total SARS Coronavirus RNA were amplified using Platinum qPCR SuperMix-UDG in 20 ml volumes with 200 nM FAM-labeled LUX primer, 200 nM reverse primer and ROX Reference Dye. PCR reactions were incubated 2 min at 50C and 2 min at 95C then cycles of: 95C. 15s; 60C. 30s on an ABI PRISM 7700 sequence detection system. The highest concentration corresponds to 5 ng total RNA.
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Serial dilutions of cDNA from total SARS Coronavirus RNA were amplified using Platinum qPCR Super Mix-UDG in 20 ml volumes with 200 nM FAM-labeled LUX primer, 200 nM reverse primer and ROX Reference Dye. PCR reactions were incubated 2 min at 50C and 2 min at 95C then cycled for 50 cylces of: 95C. 15s; 60C. 30s on an ABI PRISM 7700 sequence detection system.
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For a complete list of available primer sets please visit us at www.invitrogen.com/lux or use our database search at www.invitrogen.com/certifiedluxsearch. Product Certied LUX Primer Sets for Infectious Agents, FAM labeled Quantity 250 rxn Rxn size 20 l Cat. no. visit www.invitrogen.com/lux
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1x107 copies of qPCR Plasmid Standards, High Abundance ( Actin, B2M, GAPDH, and RPL4; Panel A) and qPCR Plasmid Standards, Medium-Low Abundance (GUS, HMBS, HPRT, and PPIA; Panel B) were amplified with Certified LUX Primer Sets labeled with FAM. All reactions were performed on the ABI PRISM 7700.
Figure 37 qPCR Plasmid Standards provide reliable normalization over a broad dynamic range
A. Amplication Plot for b-Actin using TaqMan Probes
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Panel A: TaqMan primers and probe for -Actin labeled with FAM-TAMRA used to detect 1x107 to 10 copies of the qPCR Plasmid Standards, High Abundance on the Corbett Rotor-Gene. Panel B: Certified LUX Primers for GUS labeled with FAM used to detect 1x107 to 100 copies of the qPCR Plasmid Standards, Medium-Low Abundance on the ABI PRISM 7700.
Product qPCR Plasmid Standards, High Abundance qPCR Plasmid Standards, Medium-Low Abundance
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