Two-Step qRT-HDA (Real-time quantitative RT-HDA)
The following protocols are intended for real-time detection using the Applied Biosystems 7300 Real-Time PCR System.
The kit also can be coupled with real-time detection methods to conduct real-time quantitative tHDA (qHDA) and RT-HDA (qRT-HDA) to monitor amplification as it progresses. For optimal performance, use EvaGreen as a reporter dye and ROX as a passive reference dye. Sequence-specific probes can also be designed for qHDA experiments.
- Prepare a fresh 7-fold dilution of ProtoScript RT (NEB #M0368).
- To set up a 50 μl qRT-HDA reaction, prepare a 25 μl Mix A in a 0.2 ml MicroAmp optical tube (ABI) and a 25 μl Mix B in a 0.5 ml micro centrifuge tube in a sterile hood or a PCR Workstation.
H2O X μl 10X Annealing buffer II 2.5 μl RNA template X μl Forward Primer (5 μM)* 0.75 μl Reverse Primer (5 μM)* 0.75 μl Total volume of Mix A 25 μl
H2O 8 μl 10X Annealing buffer II 2.5 μl MgSO4 (100 mM)* 1.75 μl NaCl (500 mM)* 4 μl IsoAmp® dNTP Solution 3.5 μl IsoAmp® Enzyme Mix 3.5 μl EvaGreen (20X, Biotium) 0.5 μl ROX Reference Dye (50X, Invitrogen) 1 μl ProtoScript II RT (2.0U/µl)* 0.5 μl Total volume of Mix B 25 μl
- Gently mix each of the mixes by brief vortexing or by pipetting followed by brief centrifugation. Overlay the reaction mixture with 50 μl mineral oil. Place the tubes on ice.
- Incubate Mix A at 95ºC for 2 minutes and place promptly on ice. Add 25 μl of Mix B into Mix A underneath the oil layer and gently mix the reaction by pipetting. Place the tubes on ice.
- Real-time detection is carried out on a 7300 Real-Time PCR System (ABI) with the following well inspector settings:
Reporter dye: SYBR
Passive reference dye: ROX
Use the following program:
Stage 1: (60 X) Step1: 66ºC for 0:05 Step2: 65ºC for 1:55 (Data collection and real-time analysis enabled)
Stage 2: (1 X)
Dissociation Stage (default settings of the machine; Melt curve data collection and analysis enabled)
*The condition of tHDA reactions can be further optimized by titering the following components:
Components Recommended concentration Recommended concentration for titering MgSO4 3.5 to 4 mM 3 to 4.5 mM NaCl 30 to 40 mM 20 to 50 mM Primer 75 to 100 nM 50 to 200 nM ProtoScript II RT (NEB M0368S) 1 to 2 units 0.5 to 10.5 units