SuperScript IV Reverse Transcriptase—
top IV reasons to love the best
Super RT yet
Ct
4.0
3.0
2.0
figure 5
figure 5
™
Invitrogen™ SuperScript
IV Reverse Transcriptase (RT) is the newest member of the Invitrogen™ SuperScript™
1.5
1.0
RT product line,
known for its quality and reliability in cDNA synthesis. Here are the top IV reasons why this RT
performs well.0.5
10
Degraded
Arabidopsis
RNA
Target: Gin
synthetase
Target: Gin synthetase
2.3
2
40
40
38
38
36
36
34
34
32
32
30
30
28
28
26
26
3.00
3.00
2.75
2.75
2.50
38
38
SSIII
SSIII
SSIV
SSIV
P
P
BR
BR
Q
Q
BL
BL
N
N
36
36
34
34
32
32
30
30
28
28
0 0.5 11.52
SSIII
SSIV
SSIV
P
P
T
T
BR
BR
Q
Q
BL
BL
N
N
N
0.0
RT enzyme
RT enzyme
TFRC
POLE-1
POLE-1
18S rRNA
18s rRNA
Gin
synthetase
WRKY
TF 70
GAPDH
Gin
WRKY
GAPDH
Arabidopsis
Synthetase
TF 70
Arabidopsis
EF1A
ETIF5A
EF1A
ETIF5A
Wheat germ
Wheat germ
ETIF1
ETIF1
Flax seeds
Flax seeds
Figure 2. 100-fold higher yield with degraded RNA using SuperScript IV RT. (A) A 1.2% Invitrogen™ E-Gel™ agarose gel with 500ng total RNA from
different samples. High-quality RNA (RIN >8) with intact ribosomal RNA is shown. Other RNA samples had a RIN score between 1 and 3 as determined
on an Agilent Bioanalyzer™ instrument. (B) RT-qPCR of degraded total RNA. Degraded RNA (100ng) was used in a 20μL SuperScript IV RT reaction
with random hexamers according to the provided protocol. Reverse transcriptases from other vendors were used according to the manufacturers’
recommended protocols. From each RT reaction, 10% of the cDNA was added to Invitrogen™ EXPRESS qPCR Supermix, Universal (Cat. No. 11785-01K).
Applied Biosystems™ TaqMan™ Assays for the gene targets are indicated in the figures. Ct values were normalized to SuperScript IV RT using the equation:
Normalized RT sensitivity = 2^(Ct SSIV – Ct competitor). SSIV: SuperScript IV RT. SSIII: SuperScript™ III RT.
2
18s rRNA
Gin
Synthetase
GAPDH
EF1A
ETIF5A
SSIII
P
BR
BL
4
3
SSIV
T
Q
N
4
3
1.5
TFRC
POLE-1
1
WRKY
TF 70
Arabidopsis
N
N
Input RNA
Input
RNA
Amount
Amount
SSIII
SSIV
P
N
T
Q
SSIV
P heparin
BR
Q0.5% bile
BL saltsN
0.01 U/µL
SSIV
P
BR
Q
BL
N
Reverse transcriptases
Reverse transcriptases
SSIII
SSIV
P
N
T
Q
SSIII
SSIV
P
N
T
Q
BR
SSIV
BL
Q
T
BR
8
6
0.5
TFRC
kb
SSIII
P
BR
BL
BL
SSIII
SSIV
T
Q
N
0.4
0.00
RT enzyme
SSIII
30 µM hematin
8
6
SSIII
P
BR
BL
0.20
0.25
0.00
0.00
0.0025% formalin
log10 (ng degraded RNA)
log10 (ng degraded RNA)
SSIII
SSIV
P
N
kb
BL
0.40
No inhibitors
SSIII
28
SSIV
T
Q
N
BR
Q
0.00
BL
BL
Standard
deviation
Standard
deviation
(FFPE)
RNA.
28
SSIII
P
BR
BL
T
0.6
0.2
SSIII
SSIV
P
BR
Q
SSIII
SSIV
P
BR
Q
log10 (ng degraded
RNA)
Reverse
transcriptases
log10 (ng degradedReverse
RNA) transcriptases
2.50
SSIV
T
Q
N
SSIV
0.60
P
0.20
1 ng
BL
N
N
2.25
SSIII
2.25
2.00
SSIII
Reason #4: SuperScript IV RT works every time
SSIV
2.00
1.75
SSIV
P
Inhibitory
components frequently found in RNA samples
can interfere1.75
with cDNA synthesis and give false1.50
34
P
BR
1.50
34
1.25
negative
RT-PCR and RT-qPCR results. SuperScript
IV
RT
shows
significant
resistance to contaminating
BR
1.25
Q ™
1.00
32
™
inhibitors
as compared to Invitrogen SuperScript
III RT and commercially
available RTs (Figure 4). This
Q
1.00
32
0.75
BL
0.75
0.50
30
feature
helps to obtain robust results, even with BL
lower-purity
samples
like
formalin-fixed,
paraffin-embedded
N
0.50
30
0.25
N
SSIII
P
BR
BL
0.40
0.8
Flax seeds
Wheat germ
Arabidopsis
RT
0.80
Normalized RT sensitivity
5S
SSIII
P
BR
BL
SSIII
P
BR
SSIV
BL
T
Q
N
SSIV
T
SSIII
Q
P
N
BR
BL
SSIII
P
BR
SSIV
BL
T
Q
N
SSIV
T
SSIII
Q
P
N
BR
BL
SSIII
P
BR
SSIV
BL
T
Q
N
SSIV
T
SSIII
Q
P
N
BR
BL
SSIII
P
BR
SSIV
BL
T
Q
N
SSIV
SSIIIT
Q
P
N
BR
BL
SSIII
P
BR
BL
SSIII
1.0
SSIII
Normalized RT sensitivity
5S
Wheat germ
Normalized RT sensitivity
Flax seeds
HeLa
Wheat germ
Arabidopsis
Flax seeds
HeLa
RIN
>8
Arabidopsis
RIN >8
5S
25/28S
18S
B
1.0
RIN 1-3
HeLa
RIN >8
Reason #2: SuperScript IV RT delivers up to 100x higher yield with degraded samples
The ideal RT will reverse-transcribe even the most difficult types of RNA, such as RNA samples degraded
during the purification process (which results in even fewer copies of full-length RNA transcripts).
SuperScript IV RT is robust and sensitive enough to detect degraded RNA (RIN 1–3), making it a superior
choice for cDNA synthesis.
25/28S25/28S
18S 18S
100 ng
100
ng
10 ng
10
ng
1 ng
3.00
3.00
2.75
2.75
2.50
Ct Ct
figure figure
3
3
0.60
Input RNA
Input
RNA
Amount
Amount
BR
Q
Q
BL
1.00
0.75
0.75
0.50
0.00
36
36
0.80
SSIV
P
P
BR
1.50
1.25
1.25
1.00
0 0.5 11.52
38
38
1.0
2.00
1.75
1.75
1.50
0.50
0.25
0.25
0.00
40
40
0.5
RIN 1-3
RIN 1-3
SSIII
SSIII
SSIV
Figure 3. Sensitive cDNA syntheswith a wide range of degraded plant RNA. 1–100ng of degraded Arabidopsis total RNA (RIN: 1–3) was used in a
20µL SuperScript IV RT
reactionArabidopsis
with randomRNA
hexamers according to provided protocol. Reverse transcriptasesDegraded
from otherArabidopsis
vendors were
used according
Degraded
RNA
Degraded
Arabidopsis
RNA titles.
to the manufacturers’ Degraded
recommended
protocols.
10% of cDNA in the RT reaction was added to TaqMan Assays for
targets
in 70
graph
Target:Arabidopsis
WRKY
TF 70RNA
Target:indicated
WRKY TF
Target: WRKY TF 70
Target: WRKY TF 70
1.5
A
2.50
2.25
2.25
2.00
log10 (ng degraded RNA)
log10 (ng degraded RNA)
figure 3
Variability
Degraded Degraded
Arabidopsis
RNA
Arabidopsis
RNA
Degraded
Arabidopsis
Target:Degraded
WRKY
TFGin
70RNA
Arabidopsis
RNA
Target:
synthetase
Target: WRKY
TFGin
70 synthetase
Target:
40
40
Ct Ct
Ct Ct
λ DNA/Hind III
N
Q
BR
T
P
SSIV
SSIII
3
10
Sensitivity
Degraded Arabidopsis RNA
SSIV 500ngN of Invitrogen
P ™ Ambion
T ™ Millennium™ RNA Markers (Cat. No.
Figure 1. Fast cDNA synthesis.
AM7150) was used in a 10μL SuperScript IV RT reaction with oligo(dT)20 according to the provided
protocol. Reverse
23.1 kb transcriptases from other vendors were tested following the manufacturers’
recommended 9.4
protocols, except a 10 min reaction time was used. First-strand cDNA was resolved by
6.6
alkaline gel electrophoresis and stained using Invitrogen™ SYBR™ Gold Nucleic Acid Gel Stain (Cat. No.
S11494). NaOH was used to hydrolyze all RNA, resulting in visualization of only cDNA.
6
Q
BL
BL
N
N
Reason #3: SuperScript IV RT reduces Ct by as much as 8 cycles
SuperScript IV RT has a high affinity for RNA, and due to its high processivity is capable of efficient and
log Arabidopsis
(ng degraded RNA) targets were
sensitive cDNA synthesis of any template. Triplicate qPCR reactions for two
log (ng degraded RNA)
performed; for both
targets,
SuperScript
IV
RT
resulted
in
lowest
C
values
over
all input
RNA amount (Figure 3).
Sensitivity
Variability
t
Reason #1: SuperScript IV RT reaction takes as little as 10 minutes
The more processive an enzyme, the longer the cDNA that can be synthesized and the faster it can make fulllength cDNA. Only SuperScript IV RT can synthesize up to 9kb cDNA in just 10 minutes, while the rest of the
commercially available RTs failed to synthesize cDNA up to 3kb in 30–60 minutes using their recommended
protocols (Figure 1).
9 kb
P
BR
BR
Q
32
32
30
30
28
28
26
26
Standard
deviation
Standard
deviation
BL
SS
SS
P
N
Q
T
BR
SS
SS
P
N
T
Q
SS
SS
P
N
T
Q
SS
SS
BL
BR
SS
SS
N
SS
SS
BL
8.0 kb
6.0
2
1.5
ETIF1
1
Wheat germ
Flax seeds
0.5
Figure 4. Analysis of first-strand cDNA synthesis performed in the presence of both biological and sample prep inhibitors. 500ng of Invitrogen™
Ambion™ 0.5–10kb RNA Ladder (Cat. No. 15623200) was used in a 10μL SuperScript III or SuperScript IV RT reaction with oligo(dT)20 according to the
provided protocols. Reverse transcriptases from other vendors were tested using the manufacturers’ recommended protocols. Inhibitors were added
to total RNA prior to primer annealing and addition of RT reaction mix. First-strand cDNA was resolved by alkaline gel electrophoresis and stained using
Invitrogen™ SYBR™ Gold Nucleic Acid Gel Stain (Cat. No. S11494). NaOH was used to hydrolyze all RNA, resulting in visualization of only cDNA.
100 ng
100
ng
10 ng
10
ng
1 ng
1 ng
The SuperScript IV System advantage
Invitrogen™ SuperScript™ IV Reverse Transcriptase is
available as stand-alone enzyme or in a convenient
first-strand synthesis system format. The firststrand synthesis system includes all the necessary
components for successful first-strand cDNA
synthesis in separate tubes, allowing for maximum
optimization of cDNA synthesis enabling successful
experiments.
“I tried the SuperScript IV (SSIV) RT with RNA extracted from whole blood (normal sample), plasma
(difficult sample), and laser capture samples from skin (difficult sample also). The SSIV RT performed very
well in all cases. Better than SuperScript II that I’m usually using, especially for plasma sample. It’s also
amazing because I get better results in less time!”
– Researcher, Qgenomics
Product
Size
Cat. No.
SuperScript IV Reverse Transcriptase
2000 units
18090010
10,000 units
18090050
4 x 10,000 units
18090200
50 rxns
18091050
200 rxns
18091200
SuperScript IV First-Strand Synthesis System
To find out more or to place an order, go to
thermofisher.com/ssiv
For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks
are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular
Systems, Inc., used under permission and license. Bioanalyzer is a trademark of Agilent Technologies, Inc. CO125100 0815