Spectacular is the new Super
SuperScript IV VILO Master Mix
Highest cDNA yields in the
shortest time
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
reaction
The 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).
Facts
•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
Fold change (1 ng input RNA)
Normalized fold change
=1/(2^(Ct SSIV VILO – Ct Competitor))
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
1.2
1.0
0.8
0.6
0.4
0.2
0.0
hTFRC
hB2M
hGUS
hGAPDH
hPolE
hEGF
hSTAT3
hPGK1
hPPIA
hNUF2
hANP32B
hOSTC
hEIF4E
hB2M1
Gene target
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))].
SSIV VILO linearity in TaqMan GAPDH assay
35.00
SuperScript IV VILO Master Mix
SuperScript VILO Master Mix
Supplier 1
Supplier 2
Supplier 3
Supplier 8
Supplier 9
Supplier 10
Ct
30.00
25.00
20.00
15.00
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
log [HeLa RNA input] pg
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 efficiency = 102.1%, slope = -3.3, and R2 = 0.99).
Improved performance on challenging samples
With high processivity and affinity 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 efficiency 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 efficiency among all cDNA synthesis
kits in the presence or absence of inhibitors shown.
Similarly, when SuperScript IV VILO Master Mix is tested
with RNA purified from frozen tissue samples that
commonly cause low reverse transcription efficiency,
improved performance over other cDNA synthesis kits or
master mixes is also observed (Figure 3B).
Consistently higher cDNA yields across a variety of
gene targets
To evaluate the efficiency 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.
Learn more at thermofisher.com/4vilo
A
SuperScript IV VILO Master Mix
SuperScript VILO Master MIx
Supplier 1
Supplier 2
Supplier 3
Supplier 4
Supplier 5
Supplier 6
Supplier 8
Normalized RT efficiency to SSIV VILO
1.2
1
0.8
0.6
0.4
0.2
0
No Inhibitor
75 mM LiCl
0.3% TRIzol
0.004% SDS
0.007 U/µL heparin
15 µM hemin
Inhibitor
B
SuperScript IV VILO Master Mix
SuperScript VILO Master MIx
Supplier 1
Supplier 2
Supplier 3
Supplier 11
∆Normalized RT efficiency to SSIV VILO
1.2
1
0.8
0.6
0.4
0.2
0
hTFRC
hB2M
hGUS
hGAPDH
hACTB
hEGF
TaqMan target
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.
Comparison of master mixes in TaqMan RT-qPCR gene panel analysis
Supplier 1
Supplier 2
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
-0.50
-1.00
-1.50
-2.00
-2.50
Gene name
CCDC75
MTA3
PDGFB
BCL2L1
FAR1
CXCL14
PRDX4
SGK1
TCF7
GPX4
ALAS1
ACTB
NDTCH1
MDBLKL1A
RNF38
C11
TSEN15
GC16384
OMD
TCF12
FBN1
CX3CL1
PPARGC1
FLJ4270
CAST
CMIP
LARS
TXNDC1
ZBTB10
FDXJ3
SKIL
MET
3G5H
KCNIP3
ARL1
ZIC4
MRPS24
RETSAT
KNM2B
CCDC57
NOP10
SMEK2
IGF2
NOS3
LRMP
SC4MOL
CTCF
CTNNA3
FN1
TCF3
ZNF613
ADF1
SPG7
MIF4GO
TCF4
MDM4
B2M
ADBG2
BCAR1
TRPM8
TCF25
OC28573
MAMDC2
END1
AHCTF1P
HOXA10
TLR7
GUSB
PDGFRA
TCF7L2
CTGF
APC
METTL3
RREB1
CUGBP2
MTIF2
GPR21
TBP
HPRT1
TFRC
SNF8
MLH1
DNMT3B
CCDC125
PCDH7
CCNB1
RNAdbID
ZBTB22
RPLP0
BRCA1
MARS
VEGFB
BRCA1
TFRC
REN
REN
∆Ct [=1/(2^(Ct SSIV VILO – Ct Competitor))]
SuperScript IV VILO Master Mix
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.
Integrated gDNA removal step for accurate quantification
Many RNA isolation kits and methods do not generate RNA and excludes a DNase thermal inactivation or removal
that is completely DNA-free; therefore gDNA elimination
step (Figure 5). ezDNase, a novel double-strand-specific,
remains a critical step in RT-qPCR workflow as residual
thermolabile DNase, is engineered to remove contaminating
DNA may lead to false-positive, higher background or
genomic DNA without affecting quality or quantity of target
lower assay sensitivity. The SuperScript IV VILO Master Mix RNA or damaging single-stranded DNA such as primers
with Invitrogen™ ezDNase provides a simplified workflow
and probes.
that includes a 2-minute genomic DNA elimination step
Traditional RT workflow with gDNA removal
RNA
DNase I treatment
37°C
20 min
DNase I
inactivation/+EDTA
65°C
10 min
Primer annealing
25-120
25°C
min
10
min
42°C
60 min
RT
SuperScript IV VILO workflow with ezDNase
RNA
ezDNase
treatment
37°C
2 min
Primer
annealing
25°C
10 min
SuperScript IV
VILO Master Mix
50°C
10 min
Enzyme
inactivation
85°C
5 min
Total time:
27 min
Figure 5. A timeline comparison between the use of traditional DNase I (top) and ezDNase (bottom).
Enzyme
inactivation
85°C
5 min
Total time:
>100 min
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 reactions
500 reactions
11756050
11756500
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 reactions
500 reactions
11766050
11766500
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 reactions
200 reactions
18091050
18091200
SuperScript IV Reverse Transcriptase:
Supplied with 5x RT buffer
2,000 units
10,000 units
4 x 10,000 units
18090010
18090050
18090200
50 reactions
11766051
ezDNase:
Supplied with 10x ezDNase buffer
Every step counts
Every step of your experiment is important. We are here to help you with complete solutions for every step of your
molecular biology workflow, 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 thermofisher.com/everystepcounts
Find out more at thermofisher.com/4vilo
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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