Green miRNA Two-Step qRT-PCR SuperMix

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Catalogue No: abx098036
Price: US$1,406.50
(Size: 20 rxns × 20 µl (RT Systems) / 500 rxns × 20 µl (qPCR Systems))
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Green miRNA Two-Step qRT-PCR SuperMix

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Datasheet SDS
Green miRNA Two-Step qRT-PCR SuperMix contains all the necessary components for the detection and quantification of miRNA from total RNA, small RNA and other miRNA-containing samples. The kit includes miRNA Enzyme Mix, containing Poly(A) polymerase and reverse transcriptase, and miRNA Reaction Mix to efficiently add Poly(A) tails and synthesize first-strand cDNA. Green qPCR SuperMix is provided for miRNA quantification. The Passive reference dye, which can be used to normalize fluorescent signals between ractions, is compatible with different qPCR instruments:
  • 50X Passive Reference Dye 1:ABI Prism® 7000/7300/7700/7900, ABI Step One Plus®
  • 50X Passive Reference Dye 2: ABI Prism® 7500/7500 Fast, ABI Q6, ABI QuantStudio® 6/7 Flex, ABI ViiA® 7, Stratagene Mx3000®/Mx3005P®, Qiagen Corbett Rotor-Gene® 3000.
  • No Passive Reference Dye: Roche LightCycler® 480/96, MJ Research Chromo4®, MJ Research Opticon® 2, Takara TP-800®, Bio-Rad iCycler iQ®, Bio-Rad iCycler iQ5®, Bio-Rad CFX96®, Bio-Rad CI000® Thermal Cycler, Thermo Scientific Pikoreal® 96, Qiagen Corbett Rotor-Gene® 6000/G/Q


Target Green miRNA Two-Step qRT-PCR SuperMix
Storage Store at -20°C. Stable for 12 months from date of receipt.
Kit Components
  • miRNA RT Enzyme Mix: 20 µl
  • 2X miRNA Reaction Mix: 200 µl
  • Universal miRNA qPCR Primer (10 µM): 200 µl
  • 2X Green qPCR SuperMix: 5 × 1 ml
  • 50X Passive Reference Dye (Optional): 200 µl
  • RNase-Free Water: 1 ml
Availability Shipped within 10-20 working days.
Note This product is for research use only. This product is shipped with dry ice.
Directions for use Tail addition and first strand cDNA synthesis

Reaction Components:

Component Volume
Total RNA/miRNA* x µl
miRNA RT Enzyme Mix 1 µl
2X miRNA Reaction Mix 10 µl
RNase-Free Water to 20 µl

* Total RNA should be ≤ 5 µg. Since miRNA cannot be directly quantified by the spectrophotometer, it is recommended to use 1-9 µl for a total reaction volume of 20 µl.

Mix gently, then incubate at 37 °C for 1 hour. Incubate at 85 °C for 5 seconds to inactivate the miRNA Enzyme Mix.

Suggested qPCR Conditions:

Component Volume Final Concentration
cDNA1 Variable as required
Forward Primer (10 µM)2 0.4 µl 0.2 µM
Universal miRNA qPCR Primer (10 µM) 0.4 µl 0.2 µM
2X Green qPCR SuperMix 10 µl 1X
50X Passive Reference Dye (Optional) 0.4 µl 1X
Nuclease-Free Water Variable N/A
Total Volume 20 µl N/A

1 It is recommended to dilute synthesized cDNA 5-10 fold.
2 The upstream primer is the target miRNA specific primer, which should be designed by the end user according to the target miRNA.

Thermal Cycling Conditions (2-step):

The 2-step qPCR method is recommended for higher specificity.

Number of Cycles Temperature Time
1 cycle 94 °C 30 seconds
40-45 cycles 94 °C 5 seconds
60 °C 30 seconds
Dissociation Stage

Thermal Cycling Conditions (3-step):

The 3-step qPCR method is recommended for higher sensitivity.

Number of Cycles Temperature Time
1 cycle 94 °C 30 seconds
40-45 cycles 94 °C 5 seconds
50-60 °C 15 seconds
72 °C 10 seconds
Dissociation Stage

Fluorescent signals can be collected during the annealing or extension stage. For ABI qPCR instruments, the following signal collection times are recommended:

  • ABI Prism® 7700/7900: 30 seconds
  • ABI Prism® 7000/7300: 31 seconds
  • ABI Prism® 7500: 34 seconds
  • ABI ViiA® 7: 19 seconds
Research Articles on Green miRNA Two-Step qRT-PCR SuperMix


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