Complete Solutions for Real Time PCR

Complete Solutions for Real Time PCR

• MyiQTM and iCycler iQTM Specifications

• PCR Techniques, Application examples

• Reagents and Kits

Dr. Marcus Neusser, Bio-Rad Laboratories, GermanyDr. Luis Ugozzoli, Bio-Rad Laboratories Hercules, CA

Complete Solutions for Real Time PCR

iCycler iQ® and MyiQTM

- are based on the iCyclerTM base unit with a large high resolution on-board VGA display and intuitive software

- software updates are offered by CD or internet

- 96 well reaction module for real-time PCR(various reaction modules for iCycler conventional PCR)

- 96 well reaction module with thermal gradientfor optimization of PCR in real-time PCR

- iCycler upgrade for-real time PCR with theoptical module iQ or MyiQ

iCycler

Complete Solutions for Real Time PCR

384-well

2x48-well

96-well Reaction Module

60-well

Thermal - Gradient ReactionModule

96-well Reaction Module

Thermal - Gradient ReactionModule

66.0° C65.2° C63.9° C61.8° C59.0° C57.1° C55.8° C55.0° C

back

front

[ Mg ++ ] [ Primer ] [Enzymes]

Thermal - Gradient ReactionModule

Dynamic Ramping enables that the different temperatures within the thermal gradient are reached at the same timepoint.

72 °C

95 °C

66 °C

61.8 °C

Filter Fluorescence DetectionWavelenghth specific exitation of different fluorophores and theircorresponding emission light is measuerd by a filter basedtechnology.For each fluorophore, a paired excitation and emission filter isused

The optical module iQfor real-time-PCR

CCD

Tungsten-Halogenlight source

Excitation filters96 well plate

IntensifierEmission filtersEmission light mirror

Exitation-light mirror

The MyiQ single color real-time-PCR system

MyiQ generates the same great data quality as the iQ system

Two-fold dilution series of genomic DNA

MyiQ generates the same great data quality as the iQ system

Uniformity Experiments:50 µL FAM uniformity CT = 21.41 ± 0.0950 µL SYBR uniformity CT = 19.17 ± 0.1215 µL SYBR uniformity CT = 18.71 ± 0.13

Fluorescence Detection

Sample

Sample

20 21 22

Cycle

Cycle

Sample

Fluorescence Probes

• Cleavage Based Assay - TaqMan® Assays• Displaceable Probe Assays

– molecular beacons– Dual oligo FRET probes

• Probes incorporated directly into the primers– Amplifluor™– Scorpions™

Complete Solutions for Real Time PCR

• MyiQTM and iCycler iQTM Specifications

• PCR Techniques, Application examples

• Reagents and Kits

Dr. Marcus Neusser, Bio-Rad Laboratories, GermanyPh.D. Luis Uggozzoli, Bio-Rad Laboratories CA, Herkules

4 Target Multiplex Multicolor - PCR

FAM/Tubulin

Tex Red/β-Actin

HEX/Cyclophilin

Cy5/GAPDH

Cycle

log

rela

tive

Flu

ores

cen

ce

Texas RedFAM

Hex

Cy5

10

100

1000

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49

Tubulin

Cyclophilin

beta Actin

GAPDH

Konzentration (10n)

Thre

shol

d C

ycle

Tubulin r2 = 1.000y = -3.44x + 36.82

GAPDH r2 = 0.996y = -3.34x + 37.29

β-Actin r2 = 0.998y = -3.55x + 41.07

Cyclophilin r2 = 1.000y = -3.40x + 37.04

4 Target Multiplex Multicolor - PCR

SNP Detection with FRET ProbesWild type Mutant

Heterozygote

iCycler iQ Software(Data Analysis)

PCR Quantification Demonstration ReportPCR Base Line Subtracted Data

Contains All Available Data

Current Date : Monday, June 25, 2001CurrentUser: User1Run Date: Wednesday, May 23, 2001Run User: User1

OPD File Name: beta_act1.opdOPD File Path: C:\Programme\Bio-Rad\iCycler\User1Use calibration (RME) data from OPD? Yes

Plate Setup File Name: beta_act2.ptsPlate Setup File Path: C:\Programme\Bio-Rad\iCycler\User1Protocol File Name: test.tmoProtocol File Path: C:\Programme\Bio-Rad\iCycler

Comments:beta Actin mit TaqMan Probe und mit SYBR I.Anschließende Schmelzkurvenanalyse auf Primerdimere.

Filter Wheel Setup: FilterSet4

Protocol::Protocol[]:

Standard Quantity Units: Copy Number

PCR Quantification Demonstration ReportPCR Base Line Subtracted Data

Contains All Available Data

Selected Fluorophore: FAM-490

Run Parameters

Hot Start? NoTemperature Control Mode: Algorithm Volume: 50.00 ulWell Factor Collection Mode: Experimental Plate

Data Analysis Parameters

Display ControlsPCR Display Mode (Single Point/ All Candidates): Single Point

Data SelectionPCR Reading Selection (Analysis/Collect): Analysis EnableData Window Size: 10.00%Center: end

PCR Amp/Cycle Chart

Complete Solutions for Real Time PCR

• MyiQTM and iCycler iQTM Specifications

• PCR Techniques, Application examples

• Reagents and Kits

Dr. Marcus Neusser, Bio-Rad Laboratories, GermanyPh.D. Luis Uggozzoli, Bio-Rad Laboratories CA, Herkules

Complete Solutions for Real Time PCR

Products for PCR and real-time PCR– iTaq™ DNA Polymerase– dNTPs– iQ™ Supermix, iQ™ SYBR® Green Supermix

iTaq™ SYBR Green Supermix with ROXProducts for Reverse Transcription

– iScript™ cDNA Synthesis Kit– iScript™ One-Step RT-PCR Kit with SYBR Green– iScript™ One-Step RT-PCR Kit for probes

iScript is UNIQUE

Bio-Rad’siScript cDNA Synthesis Kit

5' 3'mRNA 5'3'

First strand cDNA synthesis

Second strand cDNA synthesis

cDNA amplification

cDNA3' 5'

3'5'

5'3'

3'5'

5'3'

3'5'

5'3'

3'5'

5'3'

• RNA template• DNA primers

- gene-specific primers- oligo d(T) primers- random primers

• First Strand cDNA synthesis using Reverse Transcriptase (AMV, MMLV, Tth, etc.)

• Second strand cDNA synthesis and cDNA amplification using standard PCR conditions and enzymes

One-Step vs. Two-Step RT-PCROne-Step RT-PCR Two-Step RT-PCR

Primers + RNA

Add all reagents For RT-PCR:• reverse transcriptase• DNA polymerase• dNTPs• universal buffer

• RT (50oC, 1hr)• PCR

Primers + RNA• 70oC for 2min• Cool on ice

Add RT reagents

• RT (42oC, 1hr)

Dilute sample into new tube

Add PCR reagents

• PCR

iScript is EASY TO USE

iScript cDNA Synthesis KitTo a 0.2-ml tube add:

4 µl 5X cDNA Synthesis System1 µl iScript enzyme mixx µl nuclease-free waterx µl RNA sample20 µl final volume

OTHERTo a 0.2-ml tube add:

0.5 µl oligo(dT)12-18, 500 mg/ml0.5 µl Random hexamers, 50 ng/ml2 µl 10X buffer4 µl 25 mM MgCl21 µl 10 mM dNTP2 µl 0.1M DTT1 µl RNaseOUT (40U/µl)1 µl rt enzyme (50 U/ml)x µl DEPC-treated waterx µl RNA sample20 µl final volume

iScript is significantly FASTERiScript cDNA Synthesis Kit

5 min at 25oC 30 min at 42oC5 min at 85oCHold at 4oC

Total time: 45 minutes

With iScript – just set-up the thermal cycler and walk away!

OTHER

5 min at 65oCQuick chill on iceAdd more itemsMix and incubate for 2 min at 42oC Add RT enzymeIncubate for 50 min at 42oCHeat kill 15 min at 70oC

Total time: 85 to 90 minutes

With OTHER, you’re tied to the bench AND you’ll be waiting for your cDNAlonger!

PRODUCT PERFORMANCE DATA

Sensitivity of the iScript cDNASynthesis Kit with iTaq DNA

Polymerase for RT-PCR

M 10fg 100fg 1pg 10pg 100pg 1ng 10ng control

Reliable results across a broad range of input concentrations

iScript qRT-PCR Standard Curve Comparison: cDNA serial dilution vs. total RNA serial dilution

Log Starting Quantity (femtograms of input RNA)

Note: 1/10th of cDNA reaction used for PCR

1 2 3 4 5 6 7 8 9

ye

d u

T

5

10

15

20

25

30

35

40 cDNA Standard Total RNA Standard

cDNA total Slope -3.394 -Corr. Coef. 0.999 0.999 Intercept 38.91 3809

Complete and unbiased coverage of the RNA input population

Reagents and KitsPerformance Testing of iTaq

With Antibody Without Antibody

iScript can be used with all of Bio-Rad’s DNA polymerase products

B. iQ Supermix

C. iQ SYBR Green Supermix

A. iTaq DNA PolymeraseM 10fg 100fg 1pg 10pg 100pg 1ng 10ng control

1µg100ng10ng1ng100pg10pg1pg

Reagents and Kits iQ Supermix

Catalog # Description

170-8851 iQ ™SYBR Green Supermix w. ROX, 500 x 50 µl reactions170-8852 iQ ™SYBR Green Supermix w. ROX, 1000 x 50 µl reactions

170-8850 iQ™ SYBR Green Supermix w. ROX, 200 x 50 µl reactions170-8864 iQ ™Supermix, 1000 x 50 µl reactions170-8862 iQ ™Supermix, 500 x 50 µl reactions170-8860 iQ™ Supermix, 100 x 50 µl reactions

170-8880 iQ™ SYBR Green Supermix, 100 x 50 µl reactions170-8882 iQ ™SYBR Green Supermix, 500 x 50 µl reactions170-8884 iQ ™SYBR Green Supermix, 1000 x 50 µl reactions170-8890 iScript™ c-DNA Synthesis Kit, 25 x 20 µl reactions170-8884 iScript ™c-DNA Synthesis Kit, 100 x 20 µl reactions

www.bio-rad.com/amplification