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“ Real-time PCR ” is a variation of the standard PCR technique that is commonly used to quantify DNA or RNA in a sample. Using sequence-specific primers, the number of copies of a particular DNA or RNA sequence can be determined. By measuring the amount of amplified product at each stage during the PCR cycle, quantification is possible. If a particular sequence (DNA or RNA) is abundant in the sample, amplification is observed in earlier cycles; if the sequence is scarce, amplification is observed in later cycles. Quantification of amplified product is obtained using fluorescent probes or fluorescent DNA-binding dyes and real-time PCR instruments that measure fluorescence while performing the thermal cycling needed for the PCR reaction.
DNA polymerase
The advantages of Real-time PCR include: • Ability to monitor the progress of the PCR
Primer
reaction as it occurs in real time • Ability to precisely measure the amount of amplicon at each cycle, which allows
- Template
highly accurate quantification of the amount
- Real-time PCR primer
of starting material in samples
- DNA polymerase
• An increased dynamic range of detection
- dNTPs
• Amplification and detection occurs in a single
- Magnesium concentration
tube, eliminating post-PCR manipulations
- Reverse transcriptase - Good experimental technique
Interchelator technique [ SYBR Green method ]
Fluorescent-labeled probe technique [ Linear Probe : TaqMan method ]
Ct Area of detection for real-time PCR
Double Stranded DNA
Single Stranded
Threshold
Tm * Ct (threshold cycle) The threshold cycle (Ct) is the cycle number at which the fluorescent signal of the reaction crosses the threshold.
* Melting curve (dissociation curve) A melting curve charts the change in fluorescence observed when double-stranded DNA (dsDNA) with incorporated dye molecules dissociates, or “melts” into single-stranded DNA (ssDNA) as the temperature of the reaction is raised.
Two - step qRT-PCR Two-step qRT-PCR starts with the reverse transcription of either total RNA or poly(A)+ RNA into cDNA using a reverse transcriptase (RT). This first-strand cDNA synthesis reaction can be primed using random primers, oligo(dT), or gene-specific primers. To give an equal representation of all targets in real-time PCR applications and to avoid the 3 bias of oligo(dT) primers, many researchers use random primers or a mixture of oligo(dT) and random primers.
One – step qRT-PCR One-step qRT-PCR combines the first-strand cDNA synthesis reaction and real-time PCR reaction in the same tube, simplifying reaction setup and reducing the possibility of contamination. Gene-specific primers (GSP) are required. This is because using oligo(dT) or random primers will generate nonspecific products in the one-step procedure and reduce the amount of product of interest.
Product information
RealMODTM
RealMODTM Green
Real-time PCR Core Kit
Real-time PCR Core Kit
Cat No : 25331
Cat No : 25332
Product
RealMODTM Real-time PCR Core Kit
RealMODTM Green Real-time PCR Core Kit
Cat
25331
25332
Size
200 T
200 T
Contents
Amount
RealMODTM DNA Polymerase(5U/μl)
50 μl
10x Real-time PCR Buffer
1 ml
10x Mg2+ free Buffer
1 ml
10mM dNTP Mix(2.5mM each)
1 ml
25mM MgCl2
1 ml
RealMODTM Green DNA polymerase(5U/μl)
500 μl
10x Green PCR Buffer
1 ml
10x Mg2+ free Buffer
1 ml
10 mM dNTP Mix(2.5mM each)
1 ml
25 mM MgCl2
250 μl
HRM Dye
Experimental information
Real-time PCR analysis using the Real-MODTM Real-time Core Kit with various target genes
Real-time PCR was performed using RealMODTM Real-time PCR Core kit with various target gene. The sample was serially diluted in 10 -1 ~ 10 -7 to verify the high sensitivity.
Comparison of RealMODTM Green Real-time PCR Core Kit and Competitor A
The GAPDH DNA fragment was amplified using RealMODTM Green Real-time PCR Core Kit with SYBR Green I and HRM dye. The amplication curves were clearly observed with template gradient when HRM dye was used. With SYBR Green I, the peaks were not proportioned amount of templates.
Product information
RealMODTM Real-time PCR
RealMODTM Green Real-time PCR
Master mix Kit(2x)
Master mix Kit (2x)
Cat No : 25341 / 25342
Cat No : 25343 / 25344
Product
Cat
Size
RealMODTM Real-time PCR
25341
1 ml
Master mix Kit(2x)
25342
5 ml
RealMODTM Green Real-time
25343
1 ml
PCR Master mix Kit(2x)
25344
5 ml
Contents
2x RealMODTM PCR Solution
2x RealMODTM PCR Solution
Experimental information Real-time PCR analysis using the Real-MODTM Real-time Master mix Kit with various target genes
Synthetic RNA template(SIV NA gene, HA gene, Inf M gene and SIV NP gene) and several kinds of genomicDNA(van A, van B, van C1 gene, RNase P gene) was serially diluted (10X) resulting in 106~103 copies per sample. All reactions were performed in duplicate.
Comparison of RealMODTM Green Real-time PCR Core Kit and Competitor A
RealMODTM Green Real-time PCR Master Mix Kit (2X) is provided with HRM dye in master mix solution. To validate the storage stability of HRM dye, Real-time PCR was performed and the signal maintained the same for a year.
Product information
Label
100T
500T
RealMODTM GH Green Real-time PCR Master Mix
1 ml
1ml x 5 vials
DNase/RNase free Water
1 ml
1ml x 5 vials
Instruction Manual
1 ea
Characteristics • High Specificity : Minimize primer-dimer and non-specific amplification • High Sensitivity : Reliable detection of even low-abundance targets • Brightness : Optimum brightness for high performance on most Real-Time PCRinstruments • Hot-Start PCR : Application of antibody-based Hot-Start PCR • High reproducibility : Provides high reproducibility • Easy-to-use : Ready to perform real time PCR in the presence of a fluorescent intercalater. • Stability : Stable for over 18 months at 2~8 ºC
Experimental information RealMODTM GH Green Real-time PCR Master Mix Kit (2x) is designed for real-time PCR reaction with intercalating dye. This is a 2X concentration premix type reagents, whichcontains all reagents for real-time PCR reaction except for primers. This makes preparation of reaction mixture easer. Intercalating dye in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. This product has the effect in depression of primer dimer, which is especially important matter in intercalating dye Assay. It makes possible to measure accurate quantitative analysis in a wide range concentration of template by suppression of non-specific amplification and highly detection of a lower amount of template.
Detection of human gDNA detection with RealMOD™ GH Green Real-time PCR Master Mix Kit (2x).
Comparative Test of Dtection of human gDNA with RealMODTM GH Real-time PCR Master Mix Kit (2x) and other supplier’s kits
Product information
RealMOD™ Green FAST qPCR
RealMOD™ Green FAST qPCR
MasterMix (S)
MasterMix (LR)
Cat No : 25454
Cat No : 25453
Product RealMOD™ Green FAST qPCR MasterMix (S)
RealMOD™ Green FAST qPCR MasterMix (LR)
Cat
25454
25453
Size 5 ml (1.25ml x 4ea)
5 ml (1.25ml x 4ea)
Contents
2x RealMODTM PCR Solution
2x RealMODTM PCR Solution
Experimental information
Performance of RealMODTM Green FAST qPCR MasterMix in qPCR according to cDNA sensitivity
Performance of RealMODTM Green FAST qPCR MasterMix in qPCR according to gDNA sensitivity
Detection of Human GAPDH expression with
Detection of Human GAPDH expression with
RealMODTM
RealMODTM
Green
FAST
qPCR
MasterMix
Green
FAST
qPCR
MasterMix
Instrument : Applied Biosystems StepOnePlusTM
Instrument : Applied Biosystems StepOnePlusTM
Real-Time PCR System, Target : Human GAPDH,
Real-Time PCR System, Target : Human GAPDH,
Template : HEK293 cDNA (20ng, 4ng, 800pg,
Template : HEK293 gDNA (20ng, 4ng, 800pg,
160pg, 32pg, 6.4pg and 1.3pg)
160pg, 32pg, 6.4pg and 1.3pg)
Product information
Label
Amount
i-Green 2X qPCR Master Mix (S)
1.25 ml
qRT-PCR Enzyme Mix (50X)
40 μl
DNase/RNase Free Water
1 ml
Characteristics i-Green™ OneStep qRT-PCR Kit for RT-qPCR for fast, sensitive first-strand cDNA synthesis in gene expression analysis by real-time qPCR.
High efficiency by Hot start DNA Polymerase As all in one and premix type, it is easy to do qRT-PCR High sensitivity and signal-to-noise ratio with small amounts of RNA Outstanding reproducibility and competitively priced product
Experimental information Producing Company
Name of Machine TouchTM
Performance of i-GreenTM OneStep qRT-PCR Kit (S) in qRT-PCR according to RNA sensitivity
i-GreenTM OneStep qRT-PCR Kit (S) uses a combination of high-quality enzymes in a proprietary buffer system to deliver precise and accurate sample analysis in a high-throughput format. This kit offers ultimate convenience to the end-user in addition to guaranteed performance with respect to high sensitivity, superb signal-to noise ratio, and complete elimination of primer dimmers. This i-GreenTM OneStep qRT-PCR Kit (S) contains an i-Green 2X qPCR Master Mix (S) and qRT-PCR Enzyme Mix.
Bio-Rad Laboratories
CFX96 Real-Time PCR Detection System CFX384 TouchTM Real-Time PCR Detection System Chromo4TM Four-Color Real-Time Detector CFX ConnectTM Real-Time PCR Detection System Opticon 2 – Continuous Fluorescence Detection System MiniOpticonTM Real-Time PCR Detection System
Eppendorf
Mastercycler® ep realplex, Real-Time Thermal Cycler Mastercycler® ep realplex s, Real-Time Thermal Cycler Mastercycler® ep realplex 4, Real-Time Thermal Cycler Mastercycler® ep realplex 4s, Real-Time Thermal Cycler Mastercycler Pro, Pro S, Pro 384, Nexus Mastercycler Nexus gradient, Nexus eco, Nexus flat
Idaho Technologies
LightScanner® 24 System, 32 System RapidCycler® 2 System, RAZOR EX Instrument R.A.P.I.D. System, LT System, LT Food Security System JBAIDS System
BioGene
SynChronTM
Roche Diagnostics Ltd
LightCycler® 2.0 Instrument, 1.5 Instrument LightCycler® 480 System (system I, system II) LightCycler® 1536 System, Nano System
Qiagen
Rotor-GeneTM Q – Pure Detection Rotor-GeneTM 6000
Takara
Thermal Cycler DiceTM Real Time System
DNA-Technology
DT-96 (5 filters, same as DT prime), DTlite, DT-322
Bioer Technology
LineGene 3310/3320 Real-Time PCR Detection System LineGene K FQD-48A (M2, A4) Line Gene I & II, Line Gene 9620, 9640, 9660, 9680
Bioneer
ExicyclerTM
Corbett
Rotor Gene 3000, 6200, 62H0, 6500, 65H0, 6600
Thermo Scientific
PikoReal
Engima Diannostics
Enigma® ML
Product information
Label
Amount
i-Green 2X qPCR Master Mix (LR)
1.25 ml
qRT-PCR Enzyme Mix (50X)
40 μl
DNase/RNase Free Water
1 ml
Characteristics i-Green™ OneStep qRT-PCR Kit for RT-qPCR for fast, sensitive first-strand cDNA synthesis in gene expression analysis by real-time qPCR.
High efficiency by Hot start DNA Polymerase As all in one and premix type, it is easy to do qRT-PCR High sensitivity and signal-to-noise ratio with small amounts of RNA Outstanding reproducibility and competitively priced product
Experimental information
Performance of i-GreenTM OneStep qRT-PCR Kit (LR) in qRT-PCR according to RNA sensitivity
Producing
Name of Machine
Company
7500 Real-Time PCR System 7500 Fast Real-Time PCR System 7500 Fast Dx Real-Time PCR Instrument
Applied Biosystems 7500 Real-Time PCR System for Human Identification ViiaTM 7 Real-Time PCR System QuantStudioTM 12K Flex System
i-GreenTM OneStep qRT-PCR Kit (LR) uses a combination of high-quality enzymes in a proprietary buffer system to deliver precise and accurate sample analysis in a high-throughput format. This kit offers ultimate convenience to the end-user in addition to guaranteed performance with respect to high sensitivity, superb signal-to noise ratio, and complete elimination of primer dimmers. This i-GreenTM OneStep qRT-PCR Kit (LR) contains an i-Green 2X qPCR Master Mix (LR) and qRT-PCR Enzyme Mix.
BioGene
InSyteTM
Stratagene
Mx3000P® , Mx3005P® , Mx4000® qPCR System
Illumina
Eco Real-Time PCR System
Analytikjena
qTower, qTower 2.0, qTower 2.2
Abbott Molecular
m200RT
Product information
Contents
Amount
2X qRT-PCR Reaction Solution
0.5 ml x 1 vial
20X qRT-PCR Enzyme Mix
50 μl x 1 vial
DNase/RNase Free Water
1 ml x 1 vial
Characteristics Highest sensitivity : can use less than 0.1pg of RNA molecules Specific detection : i-StarTaq™ GH DNA polymerase with hot-start technology Reduced RNase H activity : increased specificity, higher yields, more first strand cDNA synthesis Ready to Use : Only RNA template, RNase-free water are neededEasyteps (2 tubes) Reduces reaction-to-reaction variability for improved precision in qRT-PCR Thermal stability : A half life of 100 min. at 45ºC for the highest cDNA yields from general RNA. Stability : Stable for over 2 year at -20ºC,
Experimental information High reproducibility sensitive detection of multiple RNA targets in 1 tube
HiSenScript™ RH(-) one-step qRT-PCR Kit exhibits exceptional sensitivity and an reliable quantification of up to 3 targets in a single tube by multiplex, extremely wide dynamic range in qRT-PCR assays
Suitable for real-time PCR applications
The one-Step real-time qRT-PCR with The HiSenScript™ RH(-) one-step qRT-PCR Kit is suitable for real-time PCR applications. The HiSenScript™ RH(-) one-step qRT-PCR Kit showed a superior sensitivity and a wide linear dynamic range when compared to competitor’s products
Two Step (DNA & cDNA)
Product Name
RealMOD™ Real-time PCR core Kit
RealMOD™ Green Real-time PCR core Kit
RealMOD™
RealMOD™
RealMOD™
Green
GH Green
Real-time PCR
Real-time PCR
Real-time PCR
Master Mix Kit(2X)
Master Mix Kit
Master Mix Kit
(2X)
(2x)
RealMOD™
RealMOD™
Green Fast
Green Fast
qPCR
qPCR
Master Mix (S)
Master Mix (LR)
Template
DNA & cDNA
DNA & cDNA
DNA & cDNA
DNA & cDNA
DNA & cDNA
DNA & cDNA
DNA & cDNA
Chemistry
Probe
Green dye:HRM (LC Green Plus)
Probe
Green dye:HRM (LC Green Plus)
Green dye:HRM (SYBR Green)
Green dye:HRM (EvaGreen)
Green dye:HRM (EvaGreen)
Guide
TaqMan® based detection
Application of antibody-based Hot-Start PCR
Extremely rapid qPCR in a premixed and optimized format
Extremely rapid qPCR in a premixed and optimized format
Master Mix kit
Master Mix kit
25454 (5 ml)
25453 (5 ml)
Primer Design required
Multiplex
TaqMan® based detection
Primer design required
Multiplex
Type
Core kit
Core kit
Master Mix kit
Master Mix kit
Master Mix kit
Cat. No.
25331 (200 Test)
25332 (200 Test)
25341 (1ml)
25343 (1ml)
25345 (1 ml)
25342 (5ml)
25344 (5ml)
25346 (5 ml)
One Step (RNA)
i-Green™ OneStep
i-Green™ OneStep
HiSenScript™ RH(-)
qRT-PCR Kit (LR)
qRT-PCR Kit (S)
One-Step qRT-PCR Kit
Template
RNA
RNA
RNA
Chemistry
Green dye : HRM (EvaGreen)
Green dye : HRM (EvaGreen)
Probe
Product Name
(S) : Standard (LR) : Low Rox Bio-Rad
Guide
Applied Biosystems Stratagene
Roche Eppendorf Thermo
Highest sensitivity
Specific detection
Type
Master Mix kit
Master Mix kit
Master Mix kit
Cat. No.
25107 (100 rxn)
25109 (100 rxn)
26010 (50 rxn)
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