Description
The ExcelRT™ One-Step RT-qPCR kit (TaqMan, no ROX) is designed for reverse transcription and quantitative real-time analysis of a specific target RNA by one-step reaction. The ExcelRT™ One-Step RT-qPCR kit (TaqMan, no ROX), consisting of One-Step RT Enzyme Mix and 2X One-Step qPCR Master Mix, is a convenient kit designed for highly efficient cDNA synthesis and highly specific real-time PCR in a single tube. The One-Step RT Enzyme Mix contains a thermostable ExcelRT™ Reverse Transcriptase and a RNAok™ RNase inhibitor. Consequently, One-Step RT Enzyme Mix can reverse transcribe RNA to cDNA at a wide temperature range from 42 to 60°C and active against RNase A, RNase B and RNase C. By containing specialized hot-start Taq DNA polymerase, which greatly reduce primer-dimer formation and can be activated within 2 minutes, the 2X One-Step qPCR Master Mix features high specificity and is suitable for fast cycle program.
Features
High yield
Reverse transcription at wide temperature range (42-60°C)
High specificity
Suitable for fast cycle program
With no ROX reference dye
Storage
Aliquot to avoid multiple freeze-thaw cycles (stable within 30 freeze-thaw cycles)
Protect from light
-20°C for 12 months
Reverse transcription at wide template range
ExcelRT™ One-Step RT-qPCR Kit can quantitative analyze target RNA from a wide range of RNA template input. The amplification plot of one-step RT-qPCR with total RNA templates ranging from 1 pg to 1 ng in quantity, analyzed by using RQ2110 ExcelRT™ One-Step RT-qPCR Kit (TaqMan, ROX) for RT-qPCR amplification.
Reverse transcription at wide temperature range (42-60°C)
ExcelRT™ One-Step RT-qPCR Kit can quantitative analyze target RNA at a wide temperature range (42-60°C). The amplification plot of one-step RT-qPCR with reverse transcription at temperature range from 42 to 60°C, analyzed by using RQ2110 ExcelRT™ One-Step RT-qPCR Kit for RT-qPCR amplification. The overlapped amplification curves display that ExcelRT™ One-Step RT-qPCR Kit preforms successfully cDNA synthesis at wide temperature range.
Contents
Component | Volume |
One-Step RT Enzyme Mix | 400 μl |
2X One-Step qPCR Master Mix | 2 x1 ml |
Storage
Aliquot to avoid multiple freeze-thaw cycles (stable within 30 freeze-thaw cycles)
Protect from light
-20°for 12 months
Manual
Manual_RQ2200_ExcelRT™ 2X One-Step RT-qPCR Kit (TaqMan, no ROX)
SDS
SDS_RQ2200
Recommended primer design
Amplicon size: 80-150 bp
Tm value: around 60°C (calculated with Primer3 software)
Primer length: 17-25 mer
Sequence:
‒ 45-55% of GC content isrecommended.
‒ Avoid regional high GC or ATcontent
‒ Avoid palindrome sequence
‒ Sequence with G or C at the 3" end is recommended.
Specificityof primers should be confirmed through a BLAST search.
Recommended probe design
Tm value: 6-10°C higher than primers
Probe length: 20-30 mer
Sequence:
‒ 35-65% of GC content is recommended.
‒ Avoid regional high GC or AT content
‒ Select the strand contains more C’s than G’s
‒ Avoid palindrome sequence
‒ Avoid a G at the 5" end to prevent quenching of the 5’fluorophore.
Specificityof probe should be confirmed through a BLAST search.
Recommended reaction mixture set up for qPCR
Component | Volume | Final concentration |
Template RNA | Varied | 1 pg – 1 μg |
Forward primer (10 μM) | Varied | 125 – 900 nM |
Reverse primer (10 μM) | Varied | 125 – 900 nM |
TaqMan Probe (10 μM) | Varied | 125 – 900 nM |
One-Step RT Enzyme Mix | 2 μl | 1X |
2X One-Step Master Mix | 10 μl | 1X |
ddH2O | to 20 μl | - |
Total volume | 20 µl | - |
*Template amount varies depending on the copy number of target present in the template solution.
** The PCR primer and probe concentration for an optimalqPCR reaction may vary according to primers’ and probe’s properties.
Recommended qPCRProgram
Standard program
Steps | Temp. | Time | Cycles |
Reverse transcription | 42°C - 60°C (45°C - 55°C is recommended) | 10min | 1 |
Enzyme activation | 95°C | 3 min | 1 |
Denaturation | 95°C** | 15 sec | 40-50 |
Annealing/ Extension | 60°C | 1 min |
Fast program
Steps | Temp. | Time | Cycles |
Reverse transcription | 42°C - 60°C (45°C - 55°C is recommended) | 5 min | 1 |
Enzyme activation | 95°C | 20 sec | 1 |
Denaturation | 95°C** | 3 sec | 40-50 |
Annealing/ Extension | 60°C | 30 sec |
[RP1000] ExcelRT™ Reverse Transcriptase
High yield
Thermostable, up to 50°C, during first strand synthesis
High processivity, generating cDNA up to 8 kb
Reduced RNase H ribonuclease activity
[RI1000] RNAok™ RNase Inhibitor
Application
RT-PCR
cDNA Synthesis
in vitro transcription
[RP1400] ExcelRT™ Reverse Transcription Kit II
Contains all components for reverse transcription
High yield
Thermostable, up to 50°C
Reduced RNase H ribonuclease activity
Suitable for real-time PCR
Contains all components for reverse transcription
High yield
Thermostable, up to 50°C
Reduced RNase H ribonuclease activity
Suitable for real-time PCR
[TQ2110] ExcelTaq™ 2X Q-PCR Master Mix (TaqMan, ROX)
High sensitivity
High specificity
With ROX reference dye
