Protocol for Two-step RT-qPCR using the LunaScript® RT SuperMix Kit (NEB #E3010) and the Luna® Universal qPCR Master Mix (NEB #M3003) or Luna Universal Probe qPCR Master Mix (NEB #M3004)
Step 1: First strand cDNA synthesis
1. Mix components briefly and spin down if necessary.
Note: LunaScript RT SuperMix and No-RT Control Mix are usually not frozen at -20°C.
2. Prepare cDNA synthesis reaction as described below.
|
Component |
20 μl Reaction |
Final Concentration |
|
LunaScript RT SuperMix (5X) |
4 µl |
1X |
|
RNA sample |
variable |
(up to 1 µg) |
|
Nuclease-free Water |
to 20 µl |
|
For no-RT control reactions, mix the following components.
|
Component
|
20 μl Reaction |
Final Concentration |
|
No-RT Control Mix (5X) |
4 µl |
1X |
|
RNA (up to 1 µg)* |
variable |
(up to 1 µg) |
|
Nuclease-free water |
to 20 µl |
|
*Up to 1 µg total RNA, 1 µg mRNA or 100 ng specific RNA can be used in a 20 µl reaction. However, the cDNA input for downstream qPCR detection should typically contain < 109 copies of the target to ensure that quantitation remains linear. To accommodate larger amounts of input RNA (> 1 µg), the reaction should be scaled up to ensure optimum cDNA synthesis.
3. Incubate reactions in a thermocycler with the following steps:
|
Cycle Step |
Temperature |
Time |
Cycles |
|
Primer Annealing |
25°C |
2 minutes |
1 |
|
cDNA Synthesis |
55°C |
10 minutes |
1 |
|
Heat Inactivation |
95°C |
1 minute |
1 |
Step 2: qPCR reaction
The cDNA product can be directly used in qPCR reaction. In general, 1 μl cDNA product is recommended for usage in a 20 μl qPCR detection. When necessary, up to 20% qPCR volume can be undiluted cDNA product (e.g., 4 μl cDNA product in a 20 μl qPCR reaction).
We recommend using the Luna® Universal qPCR Master Mix (NEB #M3003) and Luna Universal Probe qPCR Master Mix (NEB #M3004) for qPCR detection, as cDNA products generated using the LunaScript RT SuperMix Kit have been extensively evaluated using the two Luna kits.
- Prepare dye-based qPCR detection as follows:
|
Component |
20 μl Reaction |
Final Concentration |
|
Luna Universal qPCR Master Mix (NEB #M3003) |
10 μl |
1X |
|
10 μM forward primer |
0.5 μl |
0.25 μM |
|
10 μM reverse primer |
0.5 μl |
0.25 μM |
|
cDNA products |
1 μl |
< 4 μl |
|
Nuclease-free water |
to 20 μl |
|
Cycle Step |
Temperature |
Time |
Cycles |
|
Initial Denaturation |
95 °C |
60 seconds |
1 |
|
Denaturation |
95 °C |
15 seconds |
40-45 |
|
Melt Curve |
60–95°C |
|
1 |
- Reaction setup for probe-based qPCR detection
|
Component |
20 μl Reaction |
Final Concentration |
|
Luna Universal Probe qPCR Master Mix (NEB #M3004) |
10 μl |
1X |
|
10 μM forward primer |
0.8 μl |
0.4 μM |
|
10 μM reverse primer |
0.8 μl |
0.4 μM |
|
10 μM probe |
0.4 μl |
0.2 μM |
|
cDNA products |
1 μl |
< 4 μl |
|
Nuclease-free water |
to 20 μl |
|
|
Cycle Step |
Temperature |
Time |
Cycles |
|
Initial Denaturation |
95 °C |
60 seconds |
1 |
|
Denaturation |
95 °C |
15 seconds |
40-45 |
