Protocol for Luna® Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019)

Introduction

One-Step RT-qPCR is a convenient and powerful method for RNA detection and quantitation. The Luna Probe One-Step Reaction Mix is supplied in a convenient 1-tube format that contains all the necessary components for One-Step RT-qPCR. The 4X concentrated mix allows for a greater volume of sample input, enabling enhanced sensitivity for low titer materials (viruses, etc). It is formulated with a unique passive reference dye that is compatible across a variety of instrument platforms, including those that require a high or low ROX reference signal. The Reaction Mix also features dUTP and UDG for carryover prevention and a non-fluorescent visible dye for monitoring reaction setup. This visible dye does not overlap spectrally with fluorophores commonly used in qPCR and does not interfere with real-time detection.

Reaction Setup

  1. Thaw Luna Probe One-Step RT-qPCR 4X Mix with UDG at room temperature, then place on cold rack at 4°C or ice.

  2. After thawing completely, briefly mix all components by inversion, pipetting or gentle vortexing.

  3. Determine the total volume for the appropriate number of reactions, plus 10% overage and prepare assay mix of all components except RNA template, according to the table below.

Component

Volume per 20ul Reaction

Final Concentration

Luna Probe One-Step RT-qPCR 4X Mix with UDG

5 μL

1X

Forward Primer (10 μM)

0.8 μL

0.4 μM

Reverse Primer (10 μM)

0.8 μL

0.4 μM

Probe (10 μM)

0.4 μL

0.2 μM

Template RNA

variable

< 1 μg (total RNA)

Nuclease-free Water

to 20 μL

 

  1. Mix thoroughly but gently by pipetting or vortexing. Collect liquid to the bottom of the tube by brief centrifugation

  2. Aliquot assay mix into qPCR tubes or plate. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles.

  3. Add RNA template to qPCR tubes or plate. Seal tubes with flat, optically transparent caps; seal plates with optically transparent film. Care should be taken to properly seal plate edges and corners to prevent artifacts caused by evaporation.

  4. Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).

Programming the Real-Time Instrument

Use the All channel scan mode setting on the real-time instrument.

For faster results, the “Fast” ramp speed mode can be used where available (e.g., Applied Biosystems StepOnePlus®, QuantStudio®, 7500 Fast instruments).

CYCLE STEP

TEMPERATURE

TIME

CYCLES

 Carryover Prevention

 25°C

30 seconds

1

 Reverse Transcription

 55°C

10 minutes

 1

 Initial Denaturation

 95°C

 1 minute

 1

 Denaturation

 Extension

 95°C

 60°C

 10 seconds

 30* seconds (+plate read)

 45

*For Applied Biosystems real-time instruments (ABI), using a 60 second extension step can be beneficial for some challenging targets.

Data Analysis

  1. For basic information regarding data analysis on specific real-time PCR instruments, please refer to the user manual of the respective instrument.
  2. After the run is complete, inspect the amplification plot to ensure that the baseline threshold was set within the PCR exponential phase and above any background signal.

Additional Considerations

Notes regarding use in SARS-CoV-2 assays:

The Luna Probe One-Step RT-qPCR 4X Mix with UDG has been tested with all primer and probe sets shared by the CDC and published by the Charité Hospital (the foundation of the WHO assay). All primer/probes sets were compatible with the Luna mix. Improved performance was observed with N1, N2 (CDC) and E_Sarbeco (Charité). All three sets worked well under published primer and probe concentrations using both singleplex and multiplex conditions and with typical NEB recommended-primer and probe concentrations. Using optimal primer and probe sets, the annealing/extension can remain at 30 seconds, even if using an ABI instrument.

Please see the table below for an example of the reaction setup with SARS-CoV-2 primers/probes.

Component

Volume per 20ul Reaction

Final Concentration

Luna Probe One-Step RT-qPCR 4X Mix with UDG

5 μL

1X

SARS-CoV-2 Forward Primer (10 μM)

0.8 μL

0.4 μM

SARS-CoV-2 Reverse Primer (10 μM)

0.8 μL

0.4 μM

SARS-CoV-2 Forward Probe (10 μM)

0.4 μL

0.2 μM

Template: Test sample/Positive control/Negative control/Extraction Control

2-10 μL**

 

Nuclease-free Water

to 20 μL

 

*Primer and probe final concentrations are the same in singleplex or multiplex reactions.
**When using purified nucleic acid, we have observed tolerance of sample inputs of up to 50% of the final reaction volume.