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 reactions, 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.
Store at -20°C. Avoid repeated freeze/thaw cycles.
Shelf Life
12 months.
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. NOT FOR USE IN DIAGNOSTIC, THERAPEUTIC OR COSMETIC PROCEDURES. NOT FOR HUMAN OR ANIMAL CONSUMPTION. 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:
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