Improving cDNA yield using SuperScript™ III

Debra Ann Nickson Senior Product Manager-GenomicsqPCR Symposium – LeipzigMarch 2005

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Overview

• Researchers needs for qRT-PCR• Properties of SuperScript™ III reverse transcriptase • RNA preparation• Efficiencies of cDNA synthesis measured in two-step qRT-PCR• Choosing the best qRT-PCR product for your application

• One step vs two step• Summary

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Researchers needs for real-time qRT-PCR

• Dynamic range– Broad linear range routinely over 6-7 orders of magnitude

• Sensitivity– Low limit of detection (a few copies, sub-picogram levels of

DNA/RNA)• Specificity

– Quantify only the target of choice• Multiplexing

– Detect multiple genes in one tube, commonly for amplifying gene of interest and endogenous reference gene simultaneously

• Convenience– Closed tube assay format, mastermix configuration, amenable to

high throughput applications• Flexibility

– Compatible with various detection platforms and instrument platforms

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SuperScript™ III RT Engineering

Thumb

RNase HPalm

Fingers

Template-Primer

Three point mutations = SuperScript™ II RT

X

X

XX

Seven point mutations = SuperScript™ III RT

Fidelity: MMLV, SuperScript™ II/III, AMV: ~ 5X10-5 error rate (LacZ assay)

SuperScript™ III – the new gold standard

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RNaseH activity

AAAAAATTTTTTT

RNAse H

AA AAAATTTT

TTT

RNAse

reduces yield &

full-length cDNA

TTTTTTTAAAAAA

RNAse H

TTTTTTTAAAAAA

RNAse HH

MML-V SuperScript™ IIITotal cDNA 381ng 725ngFull length cDNA 181ng 355ng

Polymerase activity(units/mg) 330,000 410,000

Relative results using 2 µg of a 7.5kb RNA (40% which is poly A+)

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SuperScriptTM III Thermostability

6 0 0 C

0 %

2 0 %

4 0 %

6 0 %

8 0 %

1 0 0 %

0 5 1 0 1 5 2 0I n c u b a ti o n T i m e (m i n )

Rem

aini

ng R

T U

nits

(%)

S u p e r S c r ip t III

S u p e r S c r ip t II

5 0 0 C

0 %

2 0 %

4 0 %

6 0 %

8 0 %

1 0 0 %

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0I n c u b a t i o n t i m e (m i n )

Rem

aini

ng R

T U

nits

(%)

S u p e r S c r ip t III

S u p e r S c r ip t II

5 5 0 C

0 %

2 0 %

4 0 %

6 0 %

8 0 %

1 0 0 %

0 5 1 0 1 5 2 0I n c u b a ti o n T i m e (m i n )

Rem

aini

ng R

T U

nits

(%)

S u p e r S c r ip t IIIS u p e r S c r ip t IIM - M L V

50ºC

55ºC

60ºC

3.5MMLV

6.1SuperScriptTM II

220SuperScriptTM III

Half-life (min) at 50°C

Reverse Transcriptase

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SuperScript™ III high cDNA yields

SuperScript™ III Target Size

vinculin (4.6 kb)

B-factor properdin (2.4 kb)

cap binding protein (1.6 kb)

fibrillin (9.4 kb)

dynein (12.3 kb)

β-actin (353 bp)

GAPDH (532 bp)

transcription factor (3.6 kb)

guanine nuc. exchange factor (5.9 kb)

nuclear receptor coactivator (6.1 kb)

DNA polymerase ε (6.8 kb)adenomatous polyposis (8.5 kb)

Starting Total RNA (ng)

1X H

ML

1X H

ML

λ/H

indI

IIfra

gmen

ts

λ/H

indI

IIfra

gmen

ts

0.01 0.01 10 10 10 10 100 100 100 1000 1000 1000

β-actin353 bp

GAPDH532bp

CBP1.6 kb

BF2.4 kb

HTF3.6 kb

VIN4.6 kb

GNE5.9 kb

ACTR6.1 kb

Pol ε6.8 kb

APC8.5 kb

Dynein12.3 kb

FIB9.4 kbTarget

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A word on template preparation

RNA Template Preparation:• Ready to use solutions TriZOL®

• Silica-based technology PureLink™ Micro-to-Midi™ Total RNA Kit• High-Throughput Isolation PureLink™ 96 Total RNA Purification Kit

Eliminate genomic DNA contamination • RNA should be treated with DNase I Amplification grade• DNase I is then heat-inactivated by adding EDTA followed by incubation at 65ºC for 10 min.

All RNA used should have an A260/A280 ratio higher than 1.95

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Efficiencies of cDNA synthesis measured in

two-step qRT-PCR

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Comparison of two step RT-PCR Kits

0.1

1

10

0 5 10 15 20 25 30 35 40 45 50

Cycle Number

∆ R

n

■ ABI Taqman® Gold RT-PCR Kit ■ IVGN Real-Time 2 Step RT-PCR Kit B-Actin Taqman Primers 50 ng to 5 pg HeLa cDNA

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Comparison of two step RT-PCR Kits

y = -3.584(x) + 31.518R2 = 0.998

y = -3.779(x) + 34.378R2 = 0.9917

0

5

10

15

20

25

30

35

40

1 10 100 1000 10000

Starting Quantity (cDNA concentration)

Thre

shol

d Cy

cle

(Ct)

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Comparison of cDNA sensitivity competitive audit

ABI cDNA and SSIII cDNA Using ABI Mix

0.1

1

10

0 5 10 15 20 25 30 35 40 45 50

■ ABI cDNA ■ IVGN SSIII cDNABoth Using ABI PCR Mix

y = -3.705(x) + 31.811R2 = 0.9944

y = -3.645(x) + 30.566R2 = 0.9924

0

5

10

15

20

25

30

1 10 100 1000

■ ABI cDNA ■ IVGN SSIII cDNABoth With ABI PCR Mix

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Comparison of cDNA sensitivity competitive audit

ABI and SSIII cDNA Using Supermix UDG

1.00E-01

1.00E+00

1.00E+01

0 5 10 15 20 25 30 35 40 45 50

Threshold Cycle

∆Rn

■ ABI cDNA ■ IVGN SSIII cDNABoth Using SM-UDG Mix

y = -3.568(x) + 30.333R2 = 0.9948

y = -3.507(x) + 29.565R2 = 0.9989

0

5

10

15

20

25

30

35

1 10 100 1000 10000

■ ABI cDNA ■ IVGN SSIII cDNABoth Using Supermix UDG

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The best qRT-PCR product for your application

High performance, real-time quantitative RT-PCR requires high performance enzymes and systems

• SuperScript™ III Reverse Transcriptase with increased thermostability and half-life for higher cDNA yields and better specificity with gene-specific primers

• Platinum® Taq DNA Polymerase with antibody-mediated hot-start technology for higher PCR specificity and yield with room temperature setup for faster, more convenient setup, without performance loss

• Easy-to-use qPCR supermixes, one-step and two-step qRT-PCR systems

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Automatic Hot Start

PCR Assembly Initial Template Denaturation

Temperature Cycling

Inactive Taq DNA Polymerase

Fully Active Taq DNA Polymerase

96°C, 30 s - 2 min

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Reactivation of Platinum®Taq activity

Platinum®Taq is activated quicker and has more enzyme activity during the PCR

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Choice of qRT-PCR assay formats

One-Step RT-PCR• Highly defined conditions

to support RT and Taq• Requires gene specific

primer• Higher throughput (many

RNA samples)• Can use all the RNA from a

small sample• Ideal for quantification of

1 or 2 messages from a large number of RNA samples

Two-Step RT-PCR• Separate conditions for

cDNA synthesis & PCR• Flexible choice of primer

– Typically oligo(dT) and/or random hexamers are used

• Ideal for quantification of multiple genes from a limited number of RNA samples

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Flexible two step qRT-PCR kits

SuperScript™ III Platinum® Two-Step qRT-PCR KitFor LUX™ and probe based detection systemsSuperScript™ III Platinum® Two-Step qRT-PCR Kit with SYBR® GreenFor detection based on SYBR® Green

Kit Components:SuperScript™ III Enzyme Mix2x RT Reaction MixE. coli RNase HDEPC-treated water50mM MgCl220x BSA 5 mg/mlROX Reference DyePlatinum® Quantitative PCR SuperMix-UDG orPlatinum® SYBR® Green qPCR SuperMix-UDG

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Convenient detection options

SuperScript™ III Platinum® Two-Step qRT-PCR Kit with SYBR® Green

provides easy and convenient detection with SYBR® Green I dye

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Convenient detection options

Provides specific detection with LUX™ Fluorogenic Primers

SuperScript™ III Platinum® Two-Step qRT-PCR Kit

Provides sensitive detection with TaqMan® Probes

Quantitative RT-PCR from cell lysates

SuperScript™ III Platinum® CellsDirectTwo-Step qRT-PCR Kit

SuperScript™ III Platinum® CellsDirectTwo-Step qRT-PCR Kit with SYBR® Green

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Single Cell Detection

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Comparison of Standard and HTP method

Standard Method

RNA+Primer+dNTP

65 degrees for 5’

Place on ice for > 1’

Add 10x buffer, MgCl2, DTT, and RnaseOut

Mix Gently

42 degrees for 2’

Add 1ul SSIII/reaction

25 degrees 10’42 degrees 50’85 degrees 5’

Add 1ul Rnase H/reaction37 degrees 20’

HTP Protocol/MasterMix format

Combine components for first-strand synthesis:Enzyme Mix, Reaction Mix, RNA Template

25 degrees 10’42 degrees 50’70 degrees 20’

Add 1ul Rnase H/reaction 37 degrees 20’

ready to use cDNA

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Sensitive one step qRT-PCR kits

SuperScript™ III Platinum® One-Step qRT-PCR KitFor LUX™ and probe based detection systems

SuperScript™ III Platinum® One-Step qRT-PCR Kit with SYBR® GreenFor detection based on SYBR® Green

Kit Components:One-Step Enzyme Mix 2X Reaction MixROX Reference Dye 20X Bovine Serum Albumin

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One-Step qRT-PCR System

Superior sensitivity with dual-labelled probes

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Ultra Sensitive one step qRT-PCR kits

RNA UltraSense™ One-Step Quantitative RT-PCR System

Kit Components:RNA UltraSense™ Enzyme Mix RNA UltraSense™ 5 x Reaction MixROX Reference Dye 20X Bovine Serum Albumin

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Ultra Sensitive one step qRT-PCR kit

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Two step kits product summary

• Characteristics– High performance and complete kit for 2-step real-time RT-PCR, for

highly sensitive and specific detection and quanitation of RNA in gene expression profiling studies

• Applications– 2-step real-time RT-PCR, using fluorogenic detection (TaqMan, LUX™)

or SYBR® Green detection– Real-time quantitative RT-PCR results directly from cells

• Ideal for– Researchers doing 2-step qRT-PCR that want best performing reagents

for reproducibility and sensitivity – Researchers currently buying separate reagents to do 2-step qRT-PCR– Researchers that want high-through-put compatible cDNA synthesis

protocols

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One step kits product summary

• Characteristics– Easy to use; reduce contamination and human error– Optimized enzyme blend of SuperScript™III RT and Platinum® Taq DNA

Polymerase– RNA UltraSense™ 2.5 more concentrated than other supermixes

• Applications– One-step real-time RT-PCR, using fluorogenic detection (TaqMan, LUX™)

or SYBR® Green detection• Ideal for

– Researchers that want best performing reagents for reproducibility and sensitivity

– Researchers that need the highest sensitivity amplification of ultra low-abundance RNA

– Researchers currently buying separate reagents to do 2-step qRT-PCR– Researchers that want rapid, streamlined protocols

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SuperScript™ III RT Engineering

Thumb

RNase HPalm

Fingers

Template-Primer

Three point mutations = SuperScript™ II RT

X

X

XX

Seven point mutations = SuperScript™ III RT

Fidelity: MMLV, SuperScript™ II/III, AMV: ~ 5X10-5 error rate (LacZ assay)

SuperScript™ III – the new gold standard