SuperScript™ IV One-Step RT-PCR System

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USER GUIDE Pub. No. MAN0016518 Rev. A.0
Guidelines for RNA samples
Catalog No.
12594025
Size
25 reaction
∤∤This kit is optimized for use with 0.01 pg to 1 μg of total RNA, and is
Package Kit compatible with total RNA, mRNA, viral RNA, or in vitro transcribed RNA.
contents
12594100
12595025
100 reaction
25 reaction (with ezDNase™ Enzyme)
 contents ∤∤High-quality, intact RNA is essential for RT-PCR, particularly for long targets.
RNA must be devoid of RNase contamination and handled using aseptic
12595100 100 reaction (with ezDNase™ Enzyme) conditions.

Storage
∤∤Isolate total RNA with TRIzol Reagent, the PureLink RNA Mini Kit, or the
™ ™

Store all contents at –20°C (non-frost-free) MagMAX -96 Total RNA Isolation Kit.
™

conditions ∤∤Determine RNA quality using a bioanalyzer or by agarose gel electrophoresis.

Required ∤∤Template: RNA Guidelines for primers
materials ∤∤Gene specific primers (forward and reverse) ∤∤Use gene-specific primers (GSPs) with the SuperScript IV One-Step RT-
™

PCR System. Oligo(dT) or random primers are not recommended, because

Timing
∤∤Preparation time: 5 minutes nonspecific products can be generated, thereby reducing the amount of target
∤∤Total cycling time: varies with target size (1–3.5 hours) RT-PCR product.
∤∤Aoptimization
final concentration of 0.5 μM for each primer is recommended, but further
∤∤The Invitrogen SuperScript IV One-Step RT-PCR System
™ ™
may be necessary.
is designed for sensitive end-point detection and analysis of
RNA by RT-PCR. The convenient formulation enables both
∤∤Design primers that anneal to the mRNA sequence in exons on both sides of an
intron or exon/exon boundary, to allow differentiation between the amplified
cDNA synthesis and PCR amplification to be performed in a cDNA and potential contaminating genomic DNA. If this approach is not
single reaction tube using gene-specific primers. feasible, use the protocol that involves residual gDNA removal with ezDNAse™
∤∤The system can amplify RNA targets up to 14 kb in length, and enzyme.
is compatible with multiplex RT-PCR. ∤∤Ensure that primers are not self-complementary or complementary to each
∤∤due to high thermostability, processivity, and ability to
SuperScript IV™
RT Mix allows high efficiency cDNA synthesis other at the 3‘ ends.

Product synthesize cDNA efficiently from a variety of RNA samples.

∤∤PCR,
To calculate primer Tm and estimate appropriate annealing temperatures for
use the Tm calculator at thermofisher.com/tmcalculator
description ∤∤2X Platinum SuperFi RT-PCR Master Mix contains
Limited product warranty
™ ™

Platinum SuperFi DNA Polymerase which has high

™ ™

specificity, offers high yield, and is ideally suited for PCR Important licensing information
applications that require sequence accuracy. The master mix
These products may be covered by one or more Limited Use Label Licenses. By use of these
uses a buffer system optimized for both RT and PCR reactions products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
in the same tube.
∤∤isezDNase
Disclaimer: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS
™
enzyme (available separately as Cat. No. 11766051) AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE,
included with the SuperScript IV One-Step RT PCR System
™ MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS
DOCUMENT, INCLUDING YOUR USE OF IT.
with ezDNase Kits for protocols that require removal of
™

residual gDNA contamination. Corporate entity: Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1.800.955.6288
©2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo
Online Visit our product page for additional information and protocols. Fisher Scientific and its subsidiaries unless otherwise specified.
resources For support, visit thermofisher.com/support.

For Research Use Only. Not for use in diagnostic procedures.

Guidelines for cDNA synthesis and PCR
∤∤Keep all components, reaction mixes, and samples on ice.
∤∤After preparing reaction mixes, transfer them to the preheated thermal cycler and start the RT-PCR program.
∤∤The optimal conditions for RT-PCR depend on primer and target sequences.
∤∤Efficient cDNA synthesis can be accomplished by a 10-minute incubation at 45–60°C. A 50°C incubation is recommended as a general starting point.
∤∤For GC-rich or structurally complex RNA templates, increasing the cDNA synthesis incubation temperatures up to 55–60 °C may improve RT-PCR results.
∤∤Use 40 cycles of amplification for RT-PCR products ≤3 kb, and 35 cycles for RT-PCR products >3 kb.
∤∤For very low RNA input or rare targets, increasing the number of PCR cycles to 40 may improve results.
∤∤The PCR extension time varies with the size of the amplicon (recommended extension time is approximately 30 seconds per 1 kb of amplicon).
SuperScript™ IV One-Step RT-PCR System protocol
Step Action Procedure details

Step Temp Time No. cycles

Reverse transcription 45–60°C 10 min
1
RT inactivation/initial denaturation 98°C 2 min
98°C 10 s
1 Program thermal cycler Amplification 55–72°C [1]
10 s 35–40 [2]
72°C 30 s/kb
Final extension 72°C 5 min 1
[1] IMPORTANT! Use the Tm calculator at thermofisher.com/tmcalculator to determine actual annealing temperature.
[2] Use 40 cycles for short (≤3 kb) templates only.

a. Combine the following components in a 0.2-mL, nuclease-free, thin-walled PCR tube on ice.
Component Volume
2X Platinum SuperFi RT-PCR Master Mix
™ ™
25 µL
Forward primer (10 µM) 2.5 µL

Prepare RT-PCR Reverse primer (10 µM) 2.5 µL

2
reaction mix SuperScript™ IV RT Mix [1] 0.5 µL
Template RNA (0.01 pg to 1 µg total RNA) varies
Nuclease-free water to 50 µL
[1] Use all components except the SuperScript™ IV RT Mix for no RT control reactions.

b. Mix gently and ensure all the components are at the bottom of the amplification tube. Centrifuge briefly if needed.

3 Run thermal cycler Place the reaction in the pre-heated thermal cycler and run program set up in step 1.

For support, visit thermofisher.com/support.

27 July 2017 -2-
SuperScript™ IV One-Step RT-PCR System with ezDNase™ Enzyme protocol
Step Action Procedure details
Prepare a 10 µL gDNA digestion reaction mix for each RT-PCR reaction. Mix the following components in a
0.2-mL, nuclease-free, thin-walled PCR tube on ice.
Component Volume
Prepare gDNA digestion 10X ezDNase Buffer
™
1 µL
1
reaction mix ezDNase Enzyme
™
1 µL
Template RNA (0.01 pg to 1 µg total RNA) varies
Nuclease-free Water to 10 µL

a. Gently mix and incubate at 37°C for 5 minutes.

2 5 min
Digest gDNA b. (Optional) For long RT-PCR products (>3 kb) add 2 µL of 13.2 mM EDTA, then incubate at 65°C for 10 minutes.
c. Briefly centrifuge the reaction mix and place the tube on ice.

Step Temp Time No. cycles

Reverse transcription 45–60°C 10 min
1
RT inactivation/initial denaturation 98°C 2 min
98°C 10 s
3 Program thermal cycler Amplification 55–72°C [1]
10 s 35–40 [2]
72°C 30 s/kb
Final extension 72°C 5 min 1
[1] IMPORTANT! Use the Tm calculator at thermofisher.com/tmcalculator to determine actual annealing temperature.
[2] Use 40 cycles for short (≤3 kb) templates only.
a. Combine the following components in the tube on ice containing the template RNA.
Component Volume
Template RNA (from step 2) 10 µL
2X Platinum SuperFi RT-PCR Master Mix
™ ™
25 µL
Prepare RT-PCR Forward primer (10 µM) 2.5 µL
4
reaction mix Reverse primer (10 µM) 2.5 µL
SuperScript IV RT Mix
™ [1]
0.5 µL
Nuclease-free water to 50 µL
[1] Use all components except the SuperScript IV RT Mix for no RT control reactions.
™

b. Mix gently and ensure all the components are at the bottom of the amplification tube. Centrifuge briefly if needed.

5 Run thermal cycler Place the reaction in the pre-heated thermal cycler and run program set up in step 3.

For support, visit thermofisher.com/support.

27 July 2017 -3-