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))].

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hTFRC hB2M hGUS hGAPDH hPolE hEGF hSTAT3

Gene target

Fold change (1 ng input RNA)

hPGK1 hPPIA hNUF2 hANP32B hOSTC hEIF4E hB2M1

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O –

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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).

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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.

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75 mM LiCl 0.3% TRIzol 0.004% SDS 0.007 U/µL heparin 15 µM hemin

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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.

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Gene name

Comparison of master mixes in TaqMan RT-qPCR gene panel analysis

Supplier 2SuperScript IV VILO Master Mix Supplier 1

CCDC75

MTA3

PDGFB

BCL2L1

FAR1

CXC

L14

PRDX4

SGK1

TCF7

GPX4

ALAS1

ACTB

NDTC

H1

MDBLK

L1A

RNF38

C11

TSEN15

GC16384

OMD

TCF12

FBN1

CX3CL1

PPARGC1

FLJ4270

CAST

CMIP

LARS

TXNDC1

ZBTB

10FD

XJ3

SKIL

MET

3G5H

KCNIP3

ARL1

ZIC4

MRPS24

RETS

ATKNM2B

CCDC57

NOP10

SMEK2

IGF2

NOS3

LRMP

SC4M

OL

CTC

FCTN

NA3

FN1

TCF3

ZNF613

ADF1

SPG7

MIF4G

OTC

F4MDM4

B2M

ADBG2

BCAR1

TRPM8

TCF25

OC28573

MAMDC2

END1

AHCTF1P

HOXA10

TLR7

GUSB

PDGFR

ATC

F7L2

CTG

FAPC

METTL3

RREB1

CUGBP2

MTIF2

GPR21

TBP

HPRT1

TFRC

SNF8

MLH

1DNMT3B

CCDC125

PCDH7

CCNB1

RNAdbID

ZBTB

22RPLP

0BRCA1

MARS

VEGFB

BRCA1

TFRC

REN

REN

∆C

t [=

1/(2

^(C

t SS

IV V

ILO

– C

t Com

petit

or))]

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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).

Learn more at thermofi sher.com/4vilo

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. COL31178 0416

Every step counts

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

Every 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