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The world leader in serving science

林有啓 (Eugene Lin, Ph.D.)

Field Application Scientist

Introduction to Real-Time PCR & StepOne PlusTM Operation

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Real Time PCR Amplification plot features

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

0 5 10 15 20 25 30 35 40

PCR cycle number

Baseline region

Threshold

C T

No Template control

Sample

No

rmal

ised

rep

ort

er f

luo

resc

ence

(R

n)

Geometric Phase:

(Exponential Phase)

Y=N0(1+E) x =N0 2n

Lower expression level

Rn

cap

PCR vessel

thermal block

sample

lens

CT = threshold cycle: the calculated fractional cycle number at which

the PCR product crosses a threshold of detection

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Real-Time PCR detection takes place in the “exponential phase”, while traditional detection takes place at the “plateau” of the reaction.

Exponential

Linear

Plateau Gel

Low Ct

(high copy #)

High Ct

(low copy #)

PCR cycles flu

ore

sce

nt sig

na

l

101

100

10-1

Threshold

of detection

Y= N0 2n , CT 與起始濃度之對數值成反比

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定性研究

• 基因型研究

-- SNP與疾病關聯性

• 病原菌偵測

• 基因體拷貝數變異

(Copy Number Variants)

• High Resolution Melt

基因定量

• 病毒定量: HBV, HCV,HPV…

• 疾病相關基因之定量

• miRNA基因調控研究

• siRNA knock down validation

• 檢測基因轉殖食品(GMO)

• Somatic Mutation檢測

同步定量PCR之應用

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Generate Real-Time Signal Using Fluorescent

1. TaqMan® probe or TaqMan® MGB probe

chemistry: 5 Nuclease assay

2. SYBR® Green I dye chemistry

F Q

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SYBR® Green Dye

• A ‘minor groove’-binding molecule specific to the minor

groove of double-stranded DNA

• It fluoresces at an increased intensity when bound

Major

Groove

Minor Groove

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Polymerization Complete

Polymerization

SYBR Green - dsDNA minor-groove binding dye

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* Use NTC to check whether non-specific product is primer dimer

* If the non-specific product is primer dimer:

1. Optimize primer concentration

2. Re-design primer pair

SYBR® Green I Dye: Melting Curve Analysis

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TaqMan Chemistry- FRET Principal

energy transfer

hv

Excitation

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Fluorogenic 5' nuclease assay (TaqMan

chemistry)

R Q forward primer

reverse

primer

3'

3'

5'

5' 3'

5'

5'

1. Polymerisation

probe

Q 3'

3'

5'

5' 3'

5'

5'

2. Strand displacement

Q 3'

3'

5'

5' 3'

5'

5'

3. Cleavage

R

3'

5'

5' 3'

5'

5'

4. Polymerisation completed

Q R

R = Reporter

Q = Quencher

3'

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Minor Groove Binder (MGB) Small molecule that fits snugly into the minor groove of duplex DNA

Stabilizes probe annealing

Non Fluorescent Quencher (NFQ)

“Dark” quencher

Acts as energy transfer acceptor that does not emit a detectable fluorescent signal

MGB probe design uses a special algorithm in

Primer Express® Software.

All probes will be short (13-25-mers)

TaqMan® MGB/NFQ Probes

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• Highly specific

• Probe Hybridization

• Very High

• Multiplex PCR

• SNP detection

• +/- application

• Ready to use 20x primer/probe mix -

no need to optimize

• Gold standard for MAQC

• PCR efficiency 100% ±10%

• Less 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

Real-time PCR Chemistries

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Multiple

tubes

High Capacity

RNA-to-cDNA™

(P/N 4387406)

High Capacity

cDNA RT Kit

(P/N 4368814)

2hr RT

0.5- 1hr RT

Step1:

Convert RNA to cDNA

(random primers/oligo dT)

Step 2:

Amplify and quantify cDNA

(gene-specific primers)

2-step qRT-PCR

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Reverse Transcription : High Capacity RNA-to-cDNA Kit

TaqMan Chemistry SYBR Chemistry

2x TaqMan Master Mix 1x 10μl

20x Probe/primer Assay Mix 1x 1μl

Water NA

cDNA 1-100 ng 5-10μl

20μl

2x Power SYBR Master Mix 1x 10μl

F Primer optimized NA

R Primer optimized NA

Water NA

cDNA 1-100 ng 5-10μl

20μl

Real-time PCR:

PCR condition:

50℃, 2min

95 ℃, 10 min

95 ℃, 15 sec 40 cycles

60 ℃, 1min

Standard mode

2X RT Buffer 10μl

20X RT Enzyme Mix 1μl

Sample (up to 2μg) Up to 9μl

Nuclease-Free water To 20μl

SYBR Green:

- Check Primer Concentration:

~100-300nM

- Add Melt (Dissociation) Curve

PCR condition:

95 ℃, 20 sec

95 ℃, 1 sec 40 cycles

60 ℃, 20 sec

Fast mode

Preparing Real-time PCR Reactions

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Real Time PCR Applications Absolute Quantitation vs. Relative Quantitation

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Log copy number

CT v

alu

es

CTs

0 1 2 3 4 5 6 7

CT is directly proportional to log of

amount of input template

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.

絕對定量 (Absolute Quantitation)

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相對定量(Relative Quantitation)

Relative— “n-fold” difference relative to the calibrator (no units)

-- 1. △ △Ct analysis (most common)

-- 2. relative standard curve

Standard: any stock RNA or DNA containing the appropriate target

Endogenous control normalized RNA input measurement and RT-efficiency difference

(ex. 18S rRNA, GAPDH, HPRT, -actin……..)

The most powerful and widely used method

Ex. Time Course (Treatment)

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Comparative Ct Method

step 2: Normalization to calibrator sample

Ct Sample – Ct Calibrator = Ct

step 1: Normalization to endogenous control

Ct Target gene – Ct Endogenous control = Ct

step 3: use the formula

2-Ct A calibrator sample is a sample to which unknown samples

are compared (ex. untreated sample or control).

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Comparative Ct Method

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

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c-Myc GAPDH Ct Ct 2-Ct

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

ΔΔCt Calculations (Comparative Ct)

t = 0

t = 12 h

t = 24 h

t = 48 h

0

2

4

6

8

10

Re

lative

Qu

an

tity

of E

xp

ressio

n

2.0

8.0

1.0 0.1

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Applied Biosystems Primers/Probe design total solution

• Option 1: Pre-Designed TaqMan® Assay (ready-to -use)

• > 1.3 million TaqMan Gene Expression assays (for 23 species)

• > 4.5 million TaqMan SNP assays

• > 1.6 million TaqMan CNV assays

• > 15,000 TaqMan microRNA assays (miRBase release 20, 206 listed species)

• TaqMan Mutation Detection assays

• TaqMan Non-Coding RNA assays

• ………

• Option 2: Custom / Custom Plus Assay

• All-in One tube TaqMan-based Assay (20X mixture)

• Option 3: Primer Express® software

• Software for designing real-time assays

• 上機條件皆相同~~不用再花時間測試primer溫度了

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How to search AB TaqMan assay on line?

https://bioinfo.appliedbiosystems.com/genome-database/

Gene name or

RefSeq Accession…….

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TaqMan® Gene Expression Array Plates

https://www.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-gene-

expression/real-time-pcr-taqman-arrays.html

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Human Alzheimer's Disease

Human Angiogenesis

Human Apoptosis

Human Immune Response

Human Inflammation

Human Phosphodiesterase

Human Protein Kinase Pathways

Human Stem Cell Pluripotency

Mouse Alzheimer's Disease

Mouse Immune Response

Mouse Stem Cell Pluripotency

Rat Inflammation

Rat Phosphodiesterase

Human Androgens

Human Chemokines

Human CYP450 and other Oxygenases

Human Diabetes

Human Estrogens

Human Heat Shock Proteins

Human Hedgehog Pathway

Human Hypoxia

Human LDL Pathways

Human Ligand Gated Ion Channels

Human MAP Kinase Pathways

Human Neurotransmitters

Human Androgens

Human Chemokines

Human Diabetes

Human Estrogens

Human Growth Factors

Human Hematopoisis

Human NGF Pathway

Human NFkB Pathway

Human Osteogenesis

Human TGFB Pathway

Human Tumor Metastasis

Human TNFR1 Pathway

Human TNFR2 Pathway

Human WNT Pathway

Human Hox Genes

Human BMP Pathway

Human IL-2 Pathway

Human IL-9 Pathway

Human iNOS Signaling

Human IL-1 Pathway

Human IL-10 Pathway

Human Growth Factors

Human Hematopoisis

Human EGF Pathway

Human FGF Pathway

Human Notch Signaling

Human ABC Transporters

Human p53 Signaling

Human CMV & MAPK Pathways

Human PDGF Pathway

Human Cholera Infection

Human Phagocytosis of Microbes

Human Pathogenesis of ALS

Human ABC Transporters

Human p53 Signaling

Human CMV & MAPK Pathways

Human PDGF Pathway

Human Cholera Infection

Human Phagocytosis of Microbes

Human Pathogenesis of ALS

Human TNF Superfamily Pathway

Human TNFR1 Pathway

Human TNFR2 Pathway

Human Toll Comparative Pathway

Human Toll-Like Receptors Pathway

Human Transcription of mRNA

Human Transcription of rRNA

Human Transcription of tRNA

Mouse lipid regulated genes

Rat Endogenous Controls

Mouse Endogenous Controls

Pre-configured TaqMan® Gene Expression Array Plate

• Pre-configured plates for 135 different diseases, pathways, and biological processes

• 96-well

• Fast(0.1 ml) plate

• Species

• Human

• Mouse

• Rat

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Targets and Pathway Information

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Plate Layout and Assay ID

Assay ID 1 2 3 4 5 6 7 8 9 10 11 12

A Hs99999901_s1 Hs99999905_m1 Hs99999909_m1 Hs99999908_m1 Hs00153836_m1 Hs00923299_m1 Hs00155658_m1 Hs00609603_m1 Hs00174915_m1 Hs00179718_m1 Hs00357608_m1 Hs00154192_m1

B Hs00609638_m1 Hs00370078_m1 Hs00234930_m1 Hs00233470_m1 Hs00233476_m1 Hs00176144_m1 Hs00176148_m1 Hs00231733_m1 Hs00754870_s1 Hs00608098_m1 Hs00231092_m1 Hs00230938_m1

C Hs00157619_m1 Hs00357739_m1 Hs00211913_m1 Hs00193363_m1 Hs00193364_m1 Hs00192033_m1 Hs00174143_m1 Hs00174131_m1 Hs00171410_m1 Hs00170103_m1 Hs00173582_m1 Hs00173745_m1

D Hs00386448_m1 Hs00166367_m1 Hs00221445_m1 Hs00195432_m1 Hs00183425_m1 Hs00232222_m1 Hs00232068_m1 Hs00195437_m1 Hs00178579_m1 Hs00178696_m1 Hs00195441_m1 Hs00415443_m1

E Hs00427259_m1 Hs00602137_m1 Hs00177066_m1 Hs00385075_m1 Hs00765707_m1 Hs00180562_m1 Hs00178463_m1 Hs00403062_m1 Hs00234686_m1 Hs00749532_s1 Hs00293689_s1 Hs00177373_m1

F Hs00955491_gH Hs00608187_m1 Hs99999918_m1 Hs00234244_m1 Hs00234245_m1 Hs00745761_s1 Hs00610319_m1 Hs00559661_m1 Hs00234257_m1 Hs00174128_m1 Hs00167060_m1 Hs00186025_m1

G Hs01117001_m1 Hs00415315_m1 Hs00193764_m1 Hs00271352_s1 Hs00245109_m1 Hs00188614_m1 Hs00178154_m1 Hs00171132_m1 Hs00220998_m1 Hs00360274_m1 Hs00195156_m1 Hs00195300_m1

H Hs00246256_m1 Hs00764128_s1 Hs00205566_m1 Hs00179759_m1 Hs00410929_m1 Hs00224203_m1 Hs00368884_g1 Hs00369400_m1 Hs00377065_m1 Hs00766203_m1 Hs00196143_m1 Hs00170423_m1

Gene Symbol 1 2 3 4 5 6 7 8 9 10 11 12

A 18S GAPDH HPRT1 GUSB ACVR1 ACVR1B ACVR2A ACVR2B AMH AMHR2 RHOA BMP2

B BMP3 BMP4 BMP5 BMP6 BMP7 BMPR1B BMPR2 CREBBP DCN E2F4 E2F5 EP300

C FMOD FNTA GDF2 MSTN GDF9 GDF10 IFNG IL6 INHA INHBA INHBB INHBC

D LTBP1 LTBP2 LTBP3 SMAD1 SMAD2 SMAD3 SMAD4 SMAD5 SMAD6 SMAD7 SMAD9 NODAL

E PPP2CA PPP2CB MAPK1 MAPK3 RBL1 RBL2 ROCK1 BMPER TSC22D1 SKP1 SP1 MAP3K7

F TFDP1 TGFA TGFB1 TGFB2 TGFB3 LEFTY2 TGFBR1 TGFBR2 TGFBR3 TNF GDF5 LTBP4

G CUL1 CHRD BMP15 NOG ZFYVE9 TGFBRAP1 ROCK2 GDF15 GDF3 RBX1 GDF11 STUB1

H FST LEFTY1 BMP10 HIPK2 SMURF1 SMURF2 INHBE IL17F ACVR1C GDF7 MAP3K7IP1 CDH1

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Configure Custom TaqMan® Array Plate

• TaqMan® Array Plates

• Fixed and custom formats: 8, 16,

32, 48, and 96

• 96-well “plates”

• Uses inventoried assays

• 10µl reaction volume for

StepOnePlus™

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What are SNPs?

• 單核苷酸多型性（Single Nucleotide Polymorphism，簡稱SNP）指的是DNA序列上發生的單個核苷酸鹼基之間的變異

• 人類遺傳基因的各種差異，有90%都可歸因於SNP的基因差異。

• 診斷及預測致病風險評估

• 藥物基因體學及新藥的開發

• Each person has 2 copies or 2 alleles of each gene – 1 allele on each

chromosome.

• Each person receives 1 allele from each parent.

• If both alleles are the same, the person is

homozygous for that gene.

• If the alleles differ, then the person is

heterozygous for that gene.

Mom Dad

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TaqMan® SNP Genotyping Assay Overview

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G

A

Homozygous GG

Heterozygous GA Homozygous AA

Allelic Discrimination (SNP) Data

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miRNA Quantitation by Real-Time PCR

Synthetic miRNA Target

let - 7a let - 7b let - 7c let - 7d Assay

for

let - 7a Northern Blot Result

Mature

miRNA

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Individual TaqMan MicroRNA Assays

• Aligned with release 19 of the miRBase

• > 12,000 TaqMan microRNA assays • Assay include: Human,

Mouse,

Rat,

Drosophila melanogaster,

Caenorhabditis elegans,

Arabidopsis thaliana

………… 193 listed species

• 46 endogenous controls (small nuclear RNAs)

U6 snRNA, RNU6B, RNU43, RNU44, RNU48, Z30, snoRNA202………..

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

• Additional Applied Biosystems Products required to run miRNA assays

-- TaqMan MicroRNA RT kit (200 rxn, 4366596/ or 1000 rxn, 4366597)

-- TaqMan Universal PCR Master Mix

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Copy Number Variation (CNV)-基因拷貝數變異

Copy Number Variation (CNV)

• A structural genomic variant involving copy number changes in comparison to a reference genome

• Deletion or duplication events involving>1 kb of DNA. Most are <10 Kb; some rare CNVs >1 Mb

• CNVs are found in normal individuals and have also been associated with disease and other phenotypes

• 異常的DNA拷貝數變化（CNV）是許多疾病（如癌症、遺傳性疾病、心血管疾病）的一種重要分子機制.

• 傳統的方法（比如G顯帶，FISH，CGH等）存在操作繁瑣，解析度低等問 題，難以提供變異區段的具體資訊。

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Workflow of TaqMan® Copy Number Variation 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

> 1.6M Pre-Designed TaqMan Copy Number Assays available

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Determination of DNA Copy Number

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• Flexible 不需要已知拷貝數的 樣品當control

• Free 免費下載分析軟體

• Easy to use 幾分鐘內完成分析，搭配圖形化介面，輕鬆了解判讀結果

• Results with confidence value 軟體內建統計運算邏輯，提供值得信賴的結果

CopyCaller™ Software-輕鬆獲得CNV結果

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TaqMan® Mutation Detection Assays (TMDA) Somatic Mutation Detection by castPCR™ Technology

Gene List AKT1

ALK

APC

BRAF

CDKN2A

CTNNB1

EGFR

FGFR3

FLT3

GNAS

HRAS

IDH1

JAK2

KRAS

KIT

MPL

NPM1

NRAS

PDGFRA

PIK3CA

PTEN

TP53 VHL

• TMDA Product Line Summary

• Assays for 778 key mutations from 46 cancer genes

• Corresponding gene reference assays

• Wild-type assays for a subset of mutation targets

• Internal Positive Control Reagents (IPC kit)

• Mutation Detector™ Software

• Somatic mutations reported in the important

genes related to biological pathways such as

EGFR, Ras-Raf, KIT, FLT3, and PDGFRA

• High sensitivity (0.1-1%) for use with FFPE

samples and biopsies

• High specificity for generating accurate results

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TaqMan® Mutation Detection Assays (TMDA)

• Superior Sensitivity – 0.1 %

• High Specificity

• Simple and scalable workflow – 3 hrs from sample to results

Competitive Allele-Specific TaqMan® PCR - castPCR

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Mutation Detector Software

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定量PCR Primers/ Probe設計軟體

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清楚明確的 TaqMan Probe & Primer 設計規範

200 bp amplicon 500 bp amplicon

3. 定量PCR Primers/ Probe設計軟體

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2. Find Primer/Probe

1. Add DNA file or Copy & Paste

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Design Parameter

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Result

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Check Tm of Primers

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SYBR Green experiment Note

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

Log of Input

Ct va

lue

GAPDH

C-Myc

∆ Ct

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簡易三步驟!!

The StepOnePlus™ Real-Time PCR System

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StepOnePlus™ Real-Time PCR System: The Basics

• 4-color instrument

• FAM™/SYBR® Green dyes

• VIC® /JOE™ dyes

• NED™/TAMRA™ dye

• ROX™ dye

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StepOnePlus™ Real-Time PCR System: The Basics

4

● 96-Well Block

- One block, 2 speeds

–Fast cycling: 40 cycles in under 40 minutes

–Standard cycling: 40 cycles in under 2 hours

–10-30µl reaction volume

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● 樣品量多時

– P/N 4346907

MicroAmp® Fast 96-Well Reaction Plate (0.1 mL) -10 plates

– P/N 4360954

MicroAmp® Optical Adhesive Film - 25 films

StepOnePlus™ Compatible Consumables

★ Place the tray containing the tube, Load at least 16 tube

● 樣品量少時

– P/N 4358293

MicroAmp® Fast 8-Tube Strip (0.1 mL) - 125 strips

– P/N 4323032

MicroAmp® Optical 8-Cap Strip - 300 strips

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

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

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• Directly load fast optical 96-well plate into the instrument

If using fast 8-tube stripes, load the tubes with fast 96-

well tray

• Do not mark any labels on the consumables

This may increase the background signal

• Avoid bubbles when pipetting into each well

Centrifuge samples

Operation Notes

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Standalone (PC-Free)

只需輕按觸控螢幕 不需電腦連線也能上機!!

1. Start the run from the touchscreen

2. After run, download the file (.eds)

to your PC

3. Analyze your data

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Browse/New Experiments: New

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Select Experiment : Save

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58

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•插入USB, 待 icon 出現在右下角, 即可點選 Collect Results檔案會自動存到 USB 中

Standalone: 只能暫存一個檔案

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StepOneTM v2.3 Software

• 一套軟體可以符合全方位的應用 (1280x1024 pixel resoltion)

•絕對定量 Quantification - Standard Curve

• 相對定量 Quantification – Comparative Ct (△△Ct)

• 相對定量 Quantification - Relative Standard Curve

• Melting Curve Analysis

• Genotyping

• Presence/Absence

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1. Run: QuickStart

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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

2. Setup: Experiment Properties

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4.

3. Setup: Run Method

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輸入偵測的基因及使用的螢光

輸入樣品名稱

5. Setup: Plate Setup 定義基因和樣品名稱

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圈選樣品擺放位置, 再從左邊勾選樣品名稱與偵測的基因

選擇Reference Sample & Endogenous Control Gene

6. Setup: Plate Setup 決定基因和樣品位置

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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

6. Setup: Plate Setup 決定標準品位置

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6. Setup: Plate Setup 決定標準品位置 – Automatic Standard Curve Setup

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3. Analyze or Re-analyze

1.

2. Auto or Manual

4. Check Threshold

7. Analysis: Amplification Plot

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Analysis: View Well Table

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Analysis: Gene Expression

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Analysis: Standard Curve

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Analysis: Melting Curve (SYBR Green)

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Analysis: Multicomponent Plot

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Analysis: QC Summary Helps with Troubleshooting

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數據和圖形簡易輸出! 超easy~ Export to Excel, PowerPoint or save as jpeg

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StepOnePlus™ Real-Time PCR System: The Basics

● Veriflex™ Block -One block, Six Zones -The same “Better than gradient” feature from Veriti™ 96-well Thermal Cycler

*Image from Veriti

Thermal Cycler

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Red lines

to delineate

zones

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Setup: Run Method

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Useful information--中文線上課程

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