Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
Spectacular is the new SuperSuperScript IV VILO Master Mix
Highest cDNA yields in the shortest time
• Super fast—RT reaction in 10 minutes and gDNA removal in 2 minutes
• Super high yield—over 2 cycles of lowered Ct values ahead of all other reverse transcription reagents
• Super convenient one-tube reaction master mix for 2-step RT-qPCR
• Super sensitive even with low template amounts and suboptimal purity samples
Facts
Figure 1. Highest sensitivity with SuperScript IV VILO Master Mix across 14 TaqMan gene targets. RT efficiency of SuperScript IV VILO Master Mix compared with ten commercial first strand cDNA synthesis master mixes by RT-qPCR. Master mixes were used to synthesize cDNA in 20 µL RT reactions, per manufacturer instructions, using 1 ng of total HeLa RNA input. For qPCR, 1 µL of RT reactions was used in 10 µL Invitrogen™ EXPRESS qPCR SuperMix (Cat. No. 11785200) reactions with Applied Biosystems™ TaqMan™ primer and/or probes for gene targets indicated. qPCR results are shown normalized to fold change relative to SuperScript IV VILO Master Mix [=1/(2^(Ct SSIV VILO – Ct Competitor))].
0.0
0.2
0.4
0.6
0.8
1.0
1.2
hTFRC hB2M hGUS hGAPDH hPolE hEGF hSTAT3
Gene target
Fold change (1 ng input RNA)
hPGK1 hPPIA hNUF2 hANP32B hOSTC hEIF4E hB2M1
Nor
mal
ized
fold
cha
nge
=1/
(2^
(Ct S
SIV
VIL
O –
Ct C
ompe
titor
))
SuperScript IV VILO Master Mix SuperScript VILO Master Mix Supplier 1 Supplier 2 Supplier 3 Supplier 4 Supplier 5 Supplier 6 Supplier 7 Supplier 8 Supplier 9
Invitrogen™ SuperScript™ IV VILO™ Master Mix is a cDNA reaction master mix designed for two-step quantitative reverse transcription PCR (RT-qPCR). It elevates the trusted VILO™ technology to the next level with the use of the new highly processive and thermostable Invitrogen™ SuperScript™ IV Reverse Transcriptase (RT) enzyme, which allows cDNA reaction to occur at higher temperatures and in less time. The SuperScript IV RT gives the highest cDNA yields and sensitivity even with suboptimal purity or scarce templates. With the SuperScript IV VILO Master Mix, the RT-qPCR workflow is further accelerated with the extremely fast and simple gDNA removal approach. It dramatically reduces the reverse transcription protocol time and reduces variation related to possible RNA loss or damage during the conventional DNase step. SuperScript IV VILO Master Mix is your new tool to enable more efficient and reproducible RT-qPCR.
Sensitivity and perfect linearity in a 10-minute reactionThe high processivity of SuperScript IV RT enzyme in SuperScript IV VILO Master Mix allows completion of RT reaction in 10 minutes. In this short reaction time, the master mix is capable of generating higher cDNA yields compared to those obtained by lengthier competitor protocols (Figure 1)—keeping the perfect linearity typical for VILO™ products (Figure 2). We compared several different cDNA synthesis kits in fourteen different RT-qPCR assays using low starting-RNA input and found SuperScript IV VILO Master Mix produced highest efficiency results. Compared with other competitors, SuperScript IV VILO Master Mix consistently lowered Ct values by >2 cycles (Figure 4).
Figure 2. Perfect linearity and lower Ct value with SuperScript IV VILO Master Mix. SuperScript IV VILO Master Mix compared by RT-qPCR with seven commercial master mixes using 5 orders of magnitude (10 pg–1 µg) of total HeLa RNA input in 20 µL RT reactions. Linearity for Applied Biosystems™ TaqMan™ GAPDH target shown in graph corresponds to slope and R2 calculated from Ct values for each formulation as determined by qPCR in 10 µL reactions using EXPRESS qPCR SuperMix (SuperScript IV VILO Master Mix effi ciency = 102.1%, slope = -3.3, and R2 = 0.99).
15.00
20.00
25.00
30.00
35.00
1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6
Ct
log [HeLa RNA input] pg
SSIV VILO linearity in TaqMan GAPDH assay
SuperScript IV VILO Master Mix
SuperScript VILO Master Mix
Supplier 1
Supplier 8
Supplier 2
Supplier 3
Supplier 9
Supplier 10
Learn more at thermofi sher.com/4vilo
Improved performance on challenging samplesWith high processivity and affi nity to RNA, SuperScript IV RT has been shown to result in improved cDNA synthesis performance on samples that usually are considered challenging, such as degraded RNA and/or RNA containing inhibitors.
To demonstrate the effi ciency of SuperScript IV VILO Master Mix in the presence of a variety of inhibitors, SuperScript IV VILO Master Mix was compared with other commercially available cDNA synthesis kits in RT-qPCR. Ct values generated by competing kits were normalized to Ct values of SuperScript IV VILO Master Mix using the equation: Normalized Y values = [1/(2^(Ct SSIV VILO – Ct
Competitor))]. As exhibited in Figure 3A, SuperScript IV VILO had the highest effi ciency among all cDNA synthesis kits in the presence or absence of inhibitors shown.
Similarly, when SuperScript IV VILO Master Mix is tested with RNA purifi ed from frozen tissue samples that commonly cause low reverse transcription effi ciency, improved performance over other cDNA synthesis kits or master mixes is also observed (Figure 3B).
Consistently higher cDNA yields across a variety of gene targetsTo evaluate the effi ciency of SuperScript IV VILO Master Mix on a wide variety of gene targets, we compared it to other cDNA synthesis kits for RT-qPCR applications using a panel of 96 gene targets (Figure 4). ∆Ct values for SuperScript IV VILO Master Mix and competitors are depicted in the graph. For 96% of targets evaluated, SuperScript IV VILO Master Mix achieved greater than 2 Ct values lower than competitors and SuperScript VILO Master Mix, and overall generates higher cDNA yields.
Figure 3. Higher RT efficiency on challenging samples using SuperScript IV VILO Master Mix. (A) SuperScript IV VILO Master MIx was compared to eight other master mixes.using 100 ng total HeLaRNA in 20 µL RT reactions plus or minus the concentration of the inhibitors indicated. Performance in qPCR was evaluated in 10 µL reactions with TaqMan primer and/or probes for the B2M gene targets using EXPRESS qPCR SuperMix. (B) SuperScript IV VILO Master MIx was compared with five other commercial master mixes using 50 ng of RNA derived from frozen tissue RNA in 20 µL RT reactions. Performance in qPCR was evaluated in 10 µL reactions with TaqMan primer and/or probes for the six gene targets using EXPRESS qPCR SuperMix.
0
0.2
0.4
0.6
0.8
1
1.2
No Inhibitor
Inhibitor
75 mM LiCl 0.3% TRIzol 0.004% SDS 0.007 U/µL heparin 15 µM hemin
SuperScript IV VILO Master Mix SuperScript VILO Master MIx Supplier 1 Supplier 2 Supplier 8Supplier 3 Supplier 4 Supplier 5 Supplier 6
Nor
mal
ized
RT
effic
ienc
y to
SS
IV V
ILO
A
B
Figure 4. Higher cDNA yield with SuperScript IV VILO Master Mix in Applied Biosystems™ TaqMan™ RT-qPCR gene panel analysis. Comparison of SuperScript IV VILO Master Mix with two commercial master mixes in an TaqMan RT-qPCR gene panel analysis using 100 ng total HeLa RNA in 20 µL RT reaction mixture. Performance in qPCR was evaluated in 10 µL reactions with TaqMan primer and/or probes for the gene targets indicated using EXPRESS qPCR SuperMix.
-2.50-2.00-1.50-1.00-0.500.000.501.001.502.002.503.003.504.004.50
Gene name
Comparison of master mixes in TaqMan RT-qPCR gene panel analysis
Supplier 2SuperScript IV VILO Master Mix Supplier 1
CC
DC
75M
TA3
PD
GFB
BC
L2L1
FAR
1C
XCL1
4P
RD
X4S
GK
1TC
F7G
PX4
AL
AS
1A
CTB
ND
TCH
1M
DB
LKL1
AR
NF3
8C
11TS
EN
15G
C16
384
OM
DTC
F12
FBN
1C
X3C
L1P
PA
RG
C1
FLJ4
270
CA
ST
CM
IPL
AR
ST
XN
DC
1Z
BTB
10FD
XJ3
SK
ILM
ET
3G5H
KC
NIP
3A
RL1
ZIC
4M
RP
S24
RE
TSAT
KN
M2B
CC
DC
57N
OP
10S
ME
K2
IGF2
NO
S3
LRM
PS
C4M
OL
CTC
FC
TNN
A3
FN1
TCF3
ZN
F613
AD
F1S
PG
7M
IF4G
OTC
F4M
DM
4B
2MA
DB
G2
BC
AR
1TR
PM
8TC
F25
OC
2857
3M
AM
DC
2E
ND
1A
HC
TF1P
HO
XA1
0TL
R7
GU
SB
PD
GFR
ATC
F7L2
CTG
FA
PC
ME
TTL
3R
RE
B1
CU
GB
P2
MTI
F2G
PR
21TB
PH
PR
T1TF
RC
SN
F8M
LH1
DN
MT3
BC
CD
C12
5P
CD
H7
CC
NB
1R
NA
db
IDZ
BTB
22R
PLP
0B
RC
A1M
AR
SV
EG
FBB
RC
A1TF
RC
RE
NR
EN
∆C
t [=
1/(2
^(C
t SS
IV V
ILO
– C
t Com
petit
or))]
0
0.2
0.4
0.6
0.8
1
1.2
hTFRC hB2M hGUS hGAPDH hACTB hEGF
∆N
orm
aliz
ed R
T ef
ficie
ncy
to S
SIV
VIL
O
TaqMan target
Supplier 2Supplier 1 Supplier 11Supplier 3SuperScript IV VILO Master Mix SuperScript VILO Master MIx
25-120 minRNA
Traditional RT workflow with gDNA removal
SuperScript IV VILO workflow with ezDNase
DNase I treatment37°C
20 min
37°C2 min
Primer annealing RT65°C
10 min25°C
10 minEnzyme
inactivationTotal time:>100 min
42°C60 min
85°C5 min
Enzyme inactivation
Total time:27 min
85°C5 min
ezDNase treatment
Primer annealing
25°C10 min
SuperScript IV VILO Master Mix
50°C10 minRNA
DNase Iinactivation/+EDTA
Integrated gDNA removal step for accurate quantificationMany RNA isolation kits and methods do not generate RNA that is completely DNA-free; therefore gDNA elimination remains a critical step in RT-qPCR workflow as residual DNA may lead to false-positive, higher background or lower assay sensitivity. The SuperScript IV VILO Master Mix with Invitrogen™ ezDNase provides a simplified workflow that includes a 2-minute genomic DNA elimination step
and excludes a DNase thermal inactivation or removal step (Figure 5). ezDNase, a novel double-strand-specific, thermolabile DNase, is engineered to remove contaminating genomic DNA without affecting quality or quantity of target RNA or damaging single-stranded DNA such as primers and probes.
Figure 5. A timeline comparison between the use of traditional DNase I (top) and ezDNase (bottom).
For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientifi c Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientifi c and its subsidiaries unless otherwise specifi ed. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. BN04191611 0516
In the United States:For customer service, call 1-800-766-7000To fax an order, use 1-800-926-1166To order online: fishersci.com
In Canada:For customer service, call 1-800-234-7437To fax an order, use 1-800-463-2996To order online: fishersci.ca
Find out more at thermofi sher.com/4vilo
Every step countsEvery step of your experiment is important. We are here to help you with complete solutions for every step of your molecular biology workfl ow, starting from RNA isolation, into reverse transcription, PCR, thermal cycling, electrophoresis, and quantitation. Discover our innovative and high-quality products to help streamline your experiments. Learn more or get a copy of the molecular biology handbook at thermofi sher.com/everystepcounts
Ordering information
Product Size Cat. No.
SuperScript IV VILO Master Mix:Includes 5x master mix containing SuperScript IV RT, RNase inhibitor, proprietary helper protein, random primers, oligo(dT), MgCl2 and dNTPs; no-RT control master mix; DEPC-treated water
50 reactions500 reactions
1175605011756500
SuperScript IV VILO Master Mix with ezDNase:Includes 5x master mix containing SuperScript IV RT, RNase inhibitor, proprietary helper protein, random primers, oligo(dT), MgCl2 and dNTPs; no-RT control master mix; ezDNase; ezDNase buffer; DEPC-treated water
50 reactions500 reactions
1176605011766500
SuperScript IV First-Strand Synthesis System:Includes Superscript IV RT; 5x RT buffer; RNase inhibitor; Random hexamers; Oligo(dT); dNTPs; RNase H, HeLa RNA; Sense control primer; Antisense control primer; DEPC-treated water
50 reactions200 reactions
1809105018091200
SuperScript IV Reverse Transcriptase:Supplied with 5x RT buffer
2,000 units10,000 units4 x 10,000 units
180900101809005018090200
ezDNase:Supplied with 10x ezDNase buffer
50 reactions 11766051