Applied Biosystems StepOne Plus Real-Time PCR 之操作與軟體介紹

Applied Biosystems StepOne Plus Real-Time PCR 之操作與軟體介紹

曾俞槙 Jasmin Tseng

Field Application Scientist

定性研究

•

-- SNP與疾病關聯性

-- CNV與疾病關聯性

同步定量PCR之應用

基因定量

•

•

•

•

• miRNA

•

• Stem cell study

• KRAS/ BRAF

R Q forward primer

reverse primer

3'

3' 5' 3' 5'

5' 5'

1. Polymerisation

probe

R

Q

3'

3' 5' 3' 5'

5' 5'

2. Strand displacement

Q

3'

3' 5' 3' 5'

5' 5'

3. Cleavage

R

3' 5' 3' 5'

5' 5'

4. Polymerisation completed

R Q

R = Reporter Q = Quencher

3'

TaqMan® probe chemistry:

Fluorogenic 5 ′ Nuclease assay

Polymerization Complete

Polymerization

SYBR Green 1 ^®

- dsDNA minor-groove binding dye

Denaturation

PCR

• Highly specific

• Probe Hybridization

• Very High

• Mutiplex PCR

• SNP detection

• +/- application

• Ready to use 20x primer/probe mix - no need to optimize

• Gold standard for MAQC

• PCR efficiency 100% ±10%

• Non-specific

• Very High

• No Probe is required

• Screening tool

• Need to optimize PCR program

• Need to check primer-dimer info

• Need to check PCR efficiency

Specificity

Sensitivity Flexibility

Optimization

Log copy number C T values

C _T s

0 1 2 3 4 5 6 7

C is directly proportional to log of

絕對定量( Absolute Quantitation)

-主要應用於病毒量及病原菌偵測-

To determine the actual number of copies of a

target nucleic acid within a sample with statistical

confidence.

相對定量( Relative Quantitation)

To determine fold differences of a target nucleic acid in a starting material with statistical confidence.

-- 1. ^{△ △ △ △} Ct analysis (most common) Ct analysis (most common) -- -- UB2 UB2 -- 2. Relative standard curve R elative standard curve

♦ Need endogenous gene normalizes the amount of sample added Endogenous control (ex. 18S rRNA, GAPDH, β-actin……..)

♦ The most powerful and widely used method

♦ Check primer PCR efficiency first if using SYBR !!

Comparative Ct Method

step 2: Normalization to calibrator sample ΔCt Sample – ΔCt Reference = ΔΔCt

step 1: Normalization to endogenous control

Sample: Ct Target gene – Ct Endogenous control = ΔCt sample

Reference: Ct Target gene – Ct Endogenous control = ΔCt reference

step 3: use the formula

2 2 ^{- - ΔΔ ΔΔ Ct Ct}

A reference sample is a sample to which unknown samples

are compared (ex. untreated sample or control).

Comparison of the c-myc expression level in T=0, T=12, T=24, T=48 time course study

Reverse Transcription: Ex. 5 ug RNA/ 50 uL =100 ng/uL Spectrophotometer measure RNA quantity

Real Time PCR

Unknown samples( 50 ng): T=0, T=12, T=24, T=48 Calibrator

t=0 t=12 t=24 t=48 time

total RNA cDNA

total RNA cDNA

total RNA cDNA

total RNA cDNA C-myc GAPDH C-myc GAPDH C-myc GAPDH C-myc GAPDH

Ct=30.5 Ct=23.6 Ct=27 Ct=22.6

Comparative Ct Method

c-Myc GAPDH ΔC _t ΔΔC _t 2 ^-ΔΔCt

T=0 (calibrator) 25 10 15 0 1.0

T=12hr 24 10 14 -1 2.0

T=24hr 23 11 12 -3 8.0

T=48hr 28 10 18 3 0.1

ΔΔC _t Calculations (Comparative C _t )

0 2 4 6 8 10

Relative Quantity of Expression

2.0

8.0

1

t = 0 t = 12 h t = 24 h t = 48 h

0.1

Applied Biosystems永遠走在研究的最前端 miRNA: a new gene expression regulator

Mature miRNA

• Assay include: 812 Human;

583 Mouse;

349 Rat;

62 Arabidopsis thaliana 74 Caenorhabditis elegans 76 Drosophila melanogaster

• 46 endogenous controls (small nuclear RNAs)

U6, U19, U24, U38, U43, U44, U48, U49, U66, Z30………..

• Each assay contain: 1 RT primer, 1 TaqMan Assay

50x RT reaction and 150 real time reactions (20ul /rxn)

TaqMan ^® SNP Genotyping Assay Overview

G A

Allelic Discrimination (SNP) data

Homozygous GG Heterozygous GA

Homozygous AA

Source: “Global variation in copy number in the human genome.” Richard Redon, et. al.

Nature444, 444-454 (23 November 2006)

• Redon et. al. defined copy number variation (CNV) as a deletion or duplication event involving >1 kb of DNA

– An important polymorphism – ~20,000 identified CNVs

– Corresponding to >6,000 unique

regions/locus in human genome assembly – Associated with diseases or genomic disorders

such as cancer, immune diseases, and neurological disorders, etc.

• Gene dosage effects can be phenotypic

– CYP2D6 is associated with drug metabolizing phenotype

– CCL3L1 affects the susceptibility to HIV/AIDS

CNV Importance

Workflow of TaqMan® Copy Number Assays

gDNA 1 ng / µL PCR rxn

Standard TaqMan protocol

4 replicates per gDNA sample

FAM™-labeled TEST ASSAY

VIC

-labeled CONTROL ASSAY (i.e.: RNase P)

TaqMan Master Mix

1

2

3

4

qPCR

CopyCaller

CopyCaller™ Software-輕鬆獲得CNV結果

• Flexible

• Free 免費下載分析軟體

• Easy to use

•

> 1.6M Pre-Designed TaqMan Copy Number Assays available

A pair of assays:

Allele-1 assay (wildtype)

– Allele-1 specific primer (ASP*1)

– Allele-2 Specific MGB blocker (ASB2) – locus-specific TaqMan probe (LST) – locus-specific PCR primer (LSP) Allele-2 assay (mutant)

– Allele-2 specific primer (ASP*2)

– Allele-1 Specific MGB blocker (ASB1) – locus-specific TaqMan probe (LST) – locus-specific PCR primer (LSP)

A

G T

C

Allele 1 - specific PCR

Allele 2 - specific PCR

ASB2

ASP*2

LST

LSP

LSP LST

G

MGB

A

MGB

ASP*1

ASB1

castPCR Technology

castPCR: Competitive Allele-Specific TaqMan PCR

Assay Specificity

Conventional AS-PCR vs. castPCR

28.1 50

50 T

50 28.5

48.5 A

50.0 48.2

28.5 G

A T

C

28.1 50

50 T

50 28.5

48.5 A

50.0 48.2

28.5 G

A T

C

27.3 30.5

28.1 T

37.1 28.1

28.5 A

37.3 28.1

27.2 G

A T

C

27.3 30.5

28.1 T

37.1 28.1

28.5 A

37.3 28.1

27.2 G

A T

C

Conventional AS-PCR castPCR

gDNA template

Primer base at 3′ Primer base at 3′

Ave. ΔCt (Ct _mm – Ct _pm ) = 4.1 Ave. ΔCt (Ct _mm – Ct _pm ) = 21.1

gDNA template

castPCR Is Much More Specific!

Fixed Set of Taqman Mutation Detection Assays

• A fixed set of RUO assays for detecting and quantifying the mutation status

• 68 Assays:

¾ wild-type and mutant assays

• 14 KRAS mutations: codon 12, 13, and 61

• 1 BRAF mutation: codon V600

• 29 EGFR mutations: Exon 18, 19, 20, and 21

¾ One specialty assay detecting 19 deletions on EGFR Exon19

• Three Reference assays

¾ EGFR_Ref

¾ KRAS_Ref

¾ BRAF_Ref

• IPC control kit

• www.appliedbiosystems.com/KRAS

Mutation Gene

Mutation

Free Region Mutation Region with

Numerous Mutation Loci

如何設計Real Time PCR的Primers/ Probe

針對基因定量與定性的解決方案

針對基因定量與定性的解決方案

Applied Biosystems提供Primers/Probe設計的

• TaqMan SNP Genotyping Assays/ CNV Assays

•

for SNPs and Gene Expression

– All-in One tube TaqMan-based Assay

• Primer Express Software

• Ready-to-Use Assays, single tube formulation

•

• Updated Human Genome information

Custom TaqMan Assays Service

4.5

TaqMan Gene Expression Assays

> 1,100,000 個已設計及測試過的

G. gallus (Chicken)

O. sativa B. taurus (Cow)

E. caballus (Horse) A. thaliana

D. rerio (Zebrafish) M. mulatta (Rhesus Macaque)

D. melanogaster C. familiaris (Dog)

C. elegans R. norvegicus

S. scrofa (Pig) M. musculus

O. cuniculus (Rabbit) H. sapiens

How to Search ABI TaqMan Gene Expresson assay??

www.appliedbiosystems.com.tw

TaqMan® Array Gene Signature Plates

http://www3.appliedbiosystems.com/AB_Home/products/guides/PlateGuide/index.htm

Pathway Study (II):

GeneAssist™ Pathway Atlas

• Provides >350 interactive, signal

transduction, metabolic and disease state cell pathway maps

• Incorporates information form publications

& bioinformatics

• When a protein is selected, additional gene information appears along with the

recommended Silencer® select siRNAs

and TaqMan® Gene Expression Assays

定量PCR Primers/ Probe設計軟體

清楚明確的

TaqMan Probe & Primer 設計規範

200 bp amplicon 500 bp amplicon

SYBR Green experiment procedure

1. Primer conc. Optimization

– Primer Final conc. 100-300 nM

– No primer dimer or non-specific product involved

2. PCR Primer Efficiency Validation

– Sample serial dilution to run standard curve for target gene and endogenous control gene

3. Real sample run for each gene

Primer Express 3.0 Operation

Primer Express 3.0 Operation

2. Find Primer/Probe

1. Add DNA file or Copy & Paste

Result

Design Parameter

Check Tm of primers

簡易三步驟!

1. Start the run from the touchscreen 2. After run, download the file (.eds)

to your PC

3. Analyze your data

Standalone (PC-Free) Operation

StepOnePlus™ Real-Time PCR System The Basics

● 96-Well Block

- One block, 2 speeds

–Fast cycling: 40 cycles in under 40 minutes –Standard cycling: 40 cycles in under 2 hours –10-30 µl reaction volume

4

● Veriflex™ Block

-One block, Six Zones

-The same “Better than gradient” feature from Veriti™

96-well Thermal Cycler

StepOnePlus™ Real-Time PCR System

The Basics

● Supported consumables:

– P/N 4346907

Fast 96-Well Reaction Plate (0.1 mL) -10 plates – P/N 4360954

Optical Adhesive Film - 25 films – P/N 4358293

Fast 8-Tube Strip (0.1 mL) - 125 strips –P/N 4323032

Optical 8-Cap Strip - 300 strips

StepOnePlus™ Real-Time PCR System

The Basics

Note: Pressure is required to activate

The flat edge of an applicator is rubbed back-and-forth along the length of the plate with a significant downward pressure to form a complete seal on

top the wells

Downward pressure applied

in back-and-forth motions

across the top of the plate

Downward pressure applied

in back-and-forth motions

across the top of the plate

The end of an applicator is rubbed around all the outside edges of the plate with a significant downward pressure to form a complete seal around the outside wells

Downward pressure applied in small

back - and - forth motions along all the edges

Downward pressure applied in small

back - and - forth motions along all the edges

Note: Pressure is required to activate the adhesive on the optical cover

StepOnePlus ^TM Operation Notes

• • Directly load fast optical 96- Directly load fast optical 96 -well plate into the instrument well plate into the instrument 9 9 If using the fast individual tubes or 8 If using the fast individual tubes or 8 - - tube stripes, load tube stripes, load

the tube with

the tube with fast 96- fast 96 -well tray well tray

• • Save Save your data by a USB device after each run (standalone) your data by a USB device after each run (standalone)

• • Do not mark Do not mark any labels on the consumables any labels on the consumables 9 9 This may increase the background signal This may increase the background signal

• • Avoid bubbles when pipetting Avoid bubbles when pipetting into each well into each well 9 9 Centrifuge Centrifuge samples samples

• • No Screen Saving during the run No Screen Saving during the run

Standalone Operation (單機操作)

• Soft power button on the LCD touchscreen

Browse/New Experiments: New

Select Experiment : Save

StepOne Plus 機器只能暫存一個檔案

•

icon 出現在右下角, 即可點選 Collect Results

USB 中

StepOne ^TM v2.1 software 1280x1024 pixel resolution

一套軟體可以符合全方位的應用

•

Quantification - Standard Curve

•

Quantification – Comparative Ct (△△Ct)

•

Quantification - Relative Standard Curve

• Melting Curve Analysis

• Genotyping

• Presence/Absence

1. Run: QuickStart

2. Setup: Experiment Properties

a. Experiment Name 及檔案儲存位置

b.選擇 Experiment Type

c.選擇使用螢光系統

d.選擇Ramp Speed

e.選擇實驗樣品種類

3. Setup: Run Method

4.

5. Setup: Plate Setup 定義基因和樣品名稱

輸入偵測的基因及 使用的螢光

輸入樣品名稱

6-1. Setup: Plate Setup

決定基因和樣品位置

(for ddCt)

圈選樣品擺放位置, 再從左邊 勾選樣品名稱與偵測的基因

7. Analyze

Analysis : Amplification Plot

3. Analyze or Re-analyze

1.

2. Auto or Manual

How to Set the Threshold? Auto or Manual Threshold?

Manual threshold could be used to set fixed

Analysis Report

Analysis : Gene Expression

6-2. Setup: Plate Setup

決定基因和樣品位置

(for Standard curve)

圈選樣品擺放位置, 再從左邊 勾選樣品名稱與偵測的基因

●Automatic Standard Curve Setup

Analysis : Standard Curve

Analysis : Melt Curve (SYBR Green)

QC Summary Help Your Troubleshooting

數據和圖形簡易輸出! 超easy~

Export to Excel, PowerPoint or save as jpeg

Comparative Ct Study:

1. Create Study

不限樣品盤數, 但上機條件要相同, 且每盤需含endogenous control

Comparative Ct Study:

2. Add Experiment

Comparative Ct Study:

3. Add or Edit Biological Group

若有biological replicates, 請點選

[Add Biological Group]

沒有的話請跳到

4. Analyze & check threshold

Comparative Ct Study:

3. Add or Edit Biological Group

2. 圈選這個Biological Group 的 wells

3. Click >>

4. 從下拉式選單選擇另外一盤,

steps2 and 3, 直到把這

1. 輸入Biological Group Name

5. 點此 [Save & Add Another

Group], 重覆step1~4, 設定另一

Comparative Ct Study:

4. Analyze & check threshold

Auto or Manual

確認Threshold是否設在Exponential phase

5. View Gene Expression Plot by Technical Replicates

檢視

technical replicate group 的 2 ^-ddCt

Easily

choose

Endo

Controls

檢視

biological replicate group 的 2 ^-ddCt

Easily choose reference biological group

6. View Gene Expression Plot by

Biological Replicates

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隨時複習 隨時複習 real real - - time PCR time PCR 相關知識與軟體操作 相關知識與軟體操作

Http://www.appliedbiosystems.com.tw

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