[RQ2110] ExcelRT™ 2X One-Step RT-qPCR Kit (TaqMan, ROX), 200 Rxn

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Description 

The ExcelRT™ One-Step RT-qPCR kit (TaqMan, 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, 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. This master mix includes ROX reference dye for normalization of each RT-qPCR assay.


Features

  • High yield

  • Reverse transcription at wide temperature range (42-60°C)

  • High specificity 

  • Suitable for fast cycle program

  • With 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


Odoo - Sample 1 for three columns

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.


Odoo - Sample 2 for three columns

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 (A) or kit from brand N (B) 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 x 1 ml


Storage

Aliquot to avoid multiple freeze-thaw cycles (stable within 30 freeze-thaw cycles)

Protect from light

-20° for 12 months

 

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 is recommended. 

‒ Avoid regional high GC or AT content 

‒ Avoid palindrome sequence 

‒ Sequence with G or C at the 3' end is recommended. 

  • Specificity of 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. 

  • Specificity of 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 optimal qPCR reaction may vary according to primers’ and probe’s properties.

 

Recommended qPCR Program

Standard program

Steps

Temp.

Time

Cycles

Reverse transcription

42°C - 60°C 
(45°C - 55°C is recommended)

10 min

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

 

 

Odoo - Sample 1 for three columns

[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

Odoo - Sample 3 for three columns

[RI1000] RNAok™ RNase Inhibitor

Application

  • RT-PCR

  • cDNA Synthesis 

  • in vitro transcription

Odoo - Sample 2 for three columns

[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

Odoo - Sample 3 for three columns

[TQ2110] ExcelTaq™ 2X Q-PCR Master Mix (TaqMan, ROX)

  • High sensitivity

  • High specificity

  • With ROX reference dye