Score winning cDNA yields with SuperScript III RT - Invitrogen

Score winning cDNA yields
with SuperScript III RT
SuperScript III Reverse Transcriptase
SuperScript III offers:
• Higher cDNA yields
• Higher thermal stability
• Longer half-life
...than any other RT
you could use

Announcing SuperScript TM
Reverse Transcriptase
How could we possibly improve on SuperScript II Reverse Transcriptase?
After all, more researchers all over the world depend on SuperScript than any
other Reverse Transcriptase. Well, we did it. Higher thermostability. Longer half-
life. The one Reverse Transcriptase you trust is now even more reliable. Get
full-length cDNA and higher sensitivity. Perform RT at higher temperatures and
get even higher yields. Whether you’re cloning, analyzing a single gene, or monitoring expression of
every gene, SuperScript III RT will move your research forward even faster.
SuperScript TM
SuperScript III reverse transcriptase (RT) 138 , a point -
mutant of SuperScript II RT5 , provides increased thermal
stability and higher yields. Like M-MLV RT, it’s a DNA
polymerase that synthesizes a complementary DNA strand
from single-stranded DNA, RNA, or an RNA:DNA hybrid.
And like SuperScript II RT, it’s engineered to be RNase H- ,
2
improved
Table 1 – SuperScript III features and benefits
Feature Benefit
delivering high yields and full-length cDNA. Compared to
all other RTs, SuperScript III has full activity at 50˚C and
an incredibly long half-life of 220 minutes. You’ll get even
higher cDNA yields and you can work at higher
temperatures for superior performance in all your RT
experiments (Table 1).
Half-life of 220 minutes at 50˚C High cDNA yields
Thermal stability at 50˚C Reduced background with gene-specific primer
Results with RNA secondary structure
RNase H negative High cDNA yields
Full-length cDNA to 12 kb
Routine detection from as low as 1.0 pg total RNA
Increased units Can increase RT units without inhibiting subsequent PCR (1).
Use 1-10 µl of a 20 µl RT reaction for subsequent PCR.
www.invitrogen.com

Increased half-life for higher yields
The longer half-life of SuperScript III RT gives you
greater cDNA yields. At its optimal temperature of
50˚C, SuperScript III RT has a half-life of 220
minutes—that’s 35 times longer than SuperScript II
Figure 1 – Half-life of SuperScript III RT and SuperScript II RT at 50˚C
More cDNA, get the message
SuperScript III RT generates more cDNA than any
other RT. This includes cDNA from total RNA or a
specific gene. To demonstrate, we compared cDNA
yield from various RTs (Figure 2). The results speak
A
cDNA synthesis (pmol)
Figure 2 – SuperScript III RT generates the highest
cDNA yields from total RNA
1,200
1,000
800
600
400
200
0
PowerScript
StrataScript
ImProm-II
SuperScript III
SuperScript II
45°C
SuperScript III Reverse Transcriptase
and 70 times longer than M-MLV RT. The longer
half-life makes SuperScript III more robust (Figure
1) and your experiments more dynamic.
RT was incubated in 1X first-strand buffer at
50˚C. Samples were taken at time points
indicated and activity measured by unit assay.
for themselves. It’s simple: for maximized cDNA
yield in library construction, array applications, or
RT-PCR, use SuperScript III RT and make every
message count.
B
6 kb
4 kb
2 kb
M PowerScript
StrataScript
45°C
ImProm-II
SuperScript III
SuperScript II
The cDNA synthesis reactions were
performed in 20 µl with 20 µg HeLa
total RNA, 0.5 µg of oligo(dT)25 and
0.1 µl of a-32P-dCTP (10 µCi/µl).
Competitive products followed the
manufacturer’s recommended
protocols. After the reaction mixtures
were pre-incubated at 45°C for 2 min,
1 µl of each RT (400 U for SuperScript
III) was added and incubated at 45°C
for another 50 min. The reactions were
stopped by adding 10 µl of 0.5 M EDTA.
5 µl of each reaction was TCAprecipitated
and dried on GF/C filters.
A. The total cDNA synthesized was
calculated by 32P counts.
B. The rest of the samples were ethanol
precipitated and electrophoresed on
1.2% agarose gel. The dried gel was
exposed on the film for 1 hour.
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Active at higher temperatures
SuperScript III is active in a wide temperature range,
making it more useful than any other RT you could select
(Figure 3A & 3B). It’s fully active at 50˚C but can be used
4
from 45-55˚C (Figure 4). Incubation at higher temperature
is important when using gene-specific primers (Figure 5)
or tackling RNA with secondary structure (Figure 6).
More full-length, first-strand cDNA with SuperScript TM
Figure 3A – SuperScript III compared to SuperScript II and M-MLV
kb
9.5
7.5
4.4
2.4
1.4
Figure 3B – SuperScript III outperforms other RT's
6 kb
4 kb
2 kb
Figure 4 – Activity of SuperScript III RT at various temperatures
2.4 kb (BF)
SuperScript III
M 37˚ 42˚ 45˚ 50˚ 55˚
SuperScript III StrataScript PowerScript ImProm-II Omniscript
M 45˚ 50˚ 55˚ 45˚ 50˚ 55˚ 45˚ 50˚ 55˚ 45˚ 50˚ 55˚ 45˚ 50˚ 55˚
www.invitrogen.com
SuperScript II
SuperScript II
SuperScript III
M-MLV
37˚ 42˚ 45˚ 50˚ 55˚ 37˚ 42˚ 45˚ 50˚ 55˚ M
M 45˚ 50˚ 55˚ 60˚ 45˚ 50˚ 55˚ 60˚ M
Targets
9.5 kb
7.5 kb
4.4 kb
2.4 kb
1.4 kb
III RT
An autoradiograph is shown of 32 P-labeled cDNA
synthesized from a mixture of 0.25 µg 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 32 P-labeled 1 kb DNA ladder.
32 P-labeled cDNA was synthesized in 20 µl at
temperatures indicated (˚C) for 50-60 min. from a
mixture of 0.2 µg each of 1.4 kb, 2.4 kb, 4.4 kb,
7.5 kb, and 9.5 kb poly(A)-tailed RNA and 0.5 µg
oligo(dT) 25 using buffers and components
supplied with each kit. 1 µl of each RT or 400 U of
SuperScript III was added to pre-warmed reaction
mixtures (hot start). The reactions were stopped
by adding 10 µl 0.5 M EDTA. After ethanol
precipitation, the 32 P-labeled cDNA products were
loaded on 1.2% alkaline agarose gel. The dried gel
was exposed on film for 2 hours.
cDNA was synthesized in 20 µl for 50 minutes at
temperatures indicated from 500 ng HeLa total RNA
with oligo(dT) primer. 400 U SuperScript II or
SuperScript III RT was added to pre-warmed
reaction mixtures (hot start). 2 µl of cDNA was
added to 50 µl PCR reaction containing primers for
2.4 kb Human Β-Factor properdin (BF) gene.

SuperScript III Reverse Transcriptase
Increased priming specificity with gene specific primer
SuperScript III RT’s activity at higher temperatures
allows you to use gene-specific primers with a high
Tm, increasing specificity, reducing background, and
Figure 5 – Increased specificity with various genes
Dynein (12.3 kb)
CH (9.3 kb)
Pol ε (6.8 kb)
RAD (5.6 kb)
42°C 50°C 55°C
Relax: GC-rich RNA is not a problem
High temperatures relax RNA secondary structure;
temperatures where SuperScript III is active when
other RTs are not. Increasing the reaction
temperature to 55˚C melts hairpin structures,
allowing synthesis to proceed. To demonstrate,
Figure 6 – High yields with GC-rich RNA and gene specific primer
55˚C
M
512 bp
414 bp
344 bp
225 bp
199 bp
generating higher yield of your specific product. Use
SuperScript III RT at 55˚C to achieve maximum
yield with GSPs (Figure 5).
M
cDNA was synthesized in 20 µl
from 2 µg total rat brain RNA for
the Dynein 12.3 kb gene (Oligo
dT 20) or from 1 µg total HeLa
RNA for the other targets (GSP)
using 200 units of SuperScript
III, 50 min at 42°C, 50°C, or
55°C. One tenth of each reaction
was amplified with Platinum ® Taq
DNA Polymerase High Fidelity.
we performed RT-PCR on a region of 28S rRNA that
is 84% GC-rich. SuperScript III RT performed
brilliantly, processing through all regions with high
yield and low background (Figure 6).
cDNA was synthesized from 100 ng HeLa
total RNA with gene-specific primer (GSP)
and 200 U SuperScript III RT in 20 µl for
50 minutes at 55˚C. 2 µl of cDNA was
added to 50 µl PCR reaction containing
primers for regions on 28S rRNA indicated
and 1 U of Platinum® Taq DNA Polymerase
High Fidelity, at 97˚C for 2 min., then 40
cycles, at 97˚C for 30 sec., 60˚C for 30 sec.,
and 68˚C for 1 min.
www.invitrogen.com 5

Routine detection in 1.0 pg total RNA
SuperScript III RT has high sensitivity to work with
small sample sizes. You can routinely detect genes from
low inputs of total RNA down to 1.0 pg. Compared to
other RTs, SuperScript III RT not only detects genes
Starting total HeLa
RNA (pg)
6
Figure 7 – SuperScript III RT generates highest yield with low input RNA
Starting total HeLa
RNA (pg)
GAPDH (532 bp)
GAPDH (532 bp)
Clontech
PowerScript
1
42°C – 50 min
10 100
Figure 8 – Effect of RT units and cDNA volume on PCR
www.invitrogen.com
M
Qiagen
Omniscript
M
SuperScript
50 U 200 U
II
Stratagene
StrataScript 1
42°C – 60 min
10 100
from low input RNA, it also produces the highest yield
(Figure 7). Increased units or cDNA volume is not
inhibiting to PCR (Figure 8).
Promega ImProm-II
RT System
Invitrogen
SuperScript III
50°C – 50 min
1 10 100
M M
37°C – 60 min
42°C – 60 min
50°C – 50 min
1 10 100
1 10 100
1 10 100
M M M M
SuperScript III
50 U 200 U
Invitrogen
SuperScript III
cDNA was synthesized from HeLa total RNA with oligo(dT) primer using buffer supplied with each RT
and recommended conditions (400 U of SuperScript III RT). 10% of cDNA reaction was added to 50 µl
PCR reaction containing primers for 532 bp GAPDH gene and 2 U of Platinum® Taq DNA Polymerase, 40
cycles, 1 min/kb.
TSC (5.3 kb)
cDNA was synthesized from 100 ng total HeLa RNA with oligo(dT) primers using SuperScript II at 45˚C or
SuperScript III at 50˚C. One third (7 µl) of the 20 µl reaction was amplified in 50 µl with 1 unit of Platinum ®
Taq DNA Polymerase High Fidelity at 94˚C for 2 min, then 35 cycles at 94˚C for 15 sec, 55˚C for 30 sec, and
68˚C for 6 min. PCR was performed with primers for human tuberous sclerosis (TSC 5.3 kb).

Full-length cDNA, high yields
Generate full-length cDNA with high yields from mimimal
starting material for gene analysis or cloning with
SuperScript III (Figure 9). At 200 U/µl, you can set up
reactions for high-throughput or increase units to increase
Figure 9 – SuperScript III RT generates highest yield with various-sized targets
Starting total RNA (ng)
Dynein (12.3 kb)
fibrillin (9.4 kb)
adenomatous polyposis (8.5 kb)
DNA polymerase ε (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)
β-actin (353 bp)
1X
HML
λHindIII fragments
0.01
0.01 10
yield. Compared to other RTs, SuperScript III RT not only
generates the highest yields, but it was also able to generate
the 9.4 kb fibrillin target (Figure 10).
10 10 10 10 10 10 1000 1000 1000
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 50°C. 10% of cDNA reaction was added to 50 µl 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.
Figure 10 – SuperScript III RT outperforms the competition
Starting total HeLa RNA (ng)
fibrillin (9.4 kb)
vinculin (4.6 kb)
B-factor properdin (2.4 kb)
cap binding protein (1.6 kb)
1.5X
LML
Promega
ImProm-II RT System
42˚C, 60 min
10 100 100 100
Invitrogen
SuperScript Invitrogen
SuperScript II
42˚C, 50 min
10 100 100 100
Qiagen
Omniscript III RT
50˚C, 50 min
10 100 100 100
Stratagene
StrataScript RT
37˚C, 60 min
10 100 100 100
Clontech
PowerScript RT
42˚C, 60 min
10 100 100 100
RT
42˚C, 50 min
10 100 100 100
1.5X
LML
1.5X
LML
cDNA was synthesized from 10 ng HeLa total RNA for cap binding protein (1.6 kb) or 100 ng HeLa total
RNA for B-factor properdin (2.4 kb), vinculin (4.6 kb), and fibrillin (9.4 kb) with oligo(dT) primer
using buffer and recommended conditions supplied with each RT (400 U of SuperScript III RT). 10%
of the cDNA reaction was added to a 50 µl PCR reaction containing primers for each gene and
2 U Platinum® Taq DNA Polymerase or 1 U Platinum® Taq DNA Polymerase High Fidelity, 35 cycles,
1 min/kb.
λHindIII fragments
M
9.4
4.6
2.4
1.6
www.invitrogen.com 7

Easily optimize reaction conditions
You can optimize SuperScript III RT for specific
applications to ensure the best results. We provide
Table 2 – SuperScript III RT specifications
SuperScript III at 200 U/µl to allow flexibility in
experimental design (Table 2).
Units To increase yields, use 2 µl, or 400 U. Increasing units does not inhibit subsequent
PCR as with other RNase H minus RTs. Use 400 U when synthesizing long targets (≥5 kb) at high
temperature (≥ 55˚C).
cDNA Volume 1-10 µl of cDNA reaction can be used in 50 µl PCR reaction.
Temperature Even though SuperScript III RT is fully active at 50˚C, you can obtain good results at 45˚C. You
should increase the temperature to 55˚C when using a gene-specific primer or working with highly
structured RNA.
Buffer SuperScript III RT uses the same 5X first-strand buffer as SuperScript II RT,
making it easy to switch. Buffer formulation: [250 mM Tris-HCI (pH 8.3), 375 mM KCI,15 mM MgCI2] Storage As a single polypeptide, SuperScript III is very stable. It is shipped on dry ice to ensure full activity
upon arrival. While SuperScript III RT is stable at –80˚C and can be kept at 4°C overnight without
activity loss, we recommend storage at –20˚C.
Stringent quality control to ensure your results
Each lot of SuperScript III RT is stringently quality
controlled. Tests include SDS-PAGE to analyze
purity; contamination assays to confirm absence
of endodeoxyribonuclease, 3´ and 5´
Figure 11 - SuperScript III RT is RNase-free
9.5 kb
7.5 kb
4.4 kb
2.4 kb
1.4 kb
0.24 kb
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
exodeoxyribonuclease, and ribonuclease activities
(Figure 11); and cDNA synthesis of 7.5-kb target
to meet yield and length criteria.
SuperScript III Reverse Transcriptase
5 µg of 0.24-9.5 kb RNA ladder was incubated with 1000 units of
SuperScript II and III at 37˚C for 30 min followed by phenol extraction,
then analyzed on a 1.25% formaldehyde/agarose/EtBr gel. Lane 1 and
2: RNA Ladder with RNase-free water kept on ice for 30 min. Lane 3
and 4: RNA ladder with SuperScript storage buffer incubated at 37˚C
for 30 min. Lane 5 and 6: RNA ladder with 1000 units SuperScript II
incubated at 37˚C for 30 min followed by phenol extraction. Lane 7 and
8: RNA ladder with 1000 units SuperScript III incubated at 37˚C for
30 min followed by phenol extraction.
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Superior performance for every application
SuperScript III lets you work at higher
temperatures to get greater cDNA yields and
reduced background with gene-specific primers.
Whether you’re cloning, analysing a single gene or
Try SuperScript TM
III today
SuperScript III Reverse Transcriptase
studying expression profiles, SuperScript III RT will
give you overall superior performance to move your
research forward even faster. For more information,
visit www.invitrogen.com/superscriptIII today.
Maximize cDNA yield, increase specificity, and enhance the results in all your RT experiments.
Call Invitrogen and order SuperScript III RT today.
Product Quantity Cat. no.
SuperScript III Reverse Transcriptase 10,000 U 18080-044
Accessories
Product Quantity Cat. no.
100 mM dNTP Set 4 x 250 µmol 10297-117
100 mM dNTP Set 4 x 25 µmol 10297-018
100 mM dATP 25 µmol 10216-018
100 mM dCTP 25 µmol 10217-016
100 mM dGTP 25 µmol 10218-014
100 mM dTTP 25 µmol 10219-012
10 mM dNTP Mix 1ml 18427-088
10 mM dNTP Mix 2.5 µmol 18427-013
10 mM dATP 2.5 µmol 18255-018
10 mM dCTP 2.5 µmol 18252-015
10 mM dGTP 2.5 µmol 18254-011
10 mM dTTP 2.5 µmol 18253-013
Oligo (dT) 12-18 Primer 25 µg 18418-012
5, 138 Products mentioned above are subject to Limited Use Label Licenses indicated by the SuperScript numbers. Please refer to the Invitrogen web site or catalogue for
Limited Use Label Licenses corresponding to the numbers indicated.
StrataScript is a trademark of Stratagene. Omniscript is a trademark of Qiagen. PowerScript is a trademark of Clontech. ImProm-II is a trademark of Promega. SuperScript
is a trademark of Invitrogen. Platinum® is a registered trademark of Invitrogen. For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Reference: 1. Huang, L. et al. (2000) Focus® 22:6.
www.invitrogen.com 9

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