Protocol for LyoPrime Luna One-Step RT-qPCR Mix with UDG (NEB #L4001S)

Introduction

One-Step RT-qPCR is a convenient and powerful method for RNA detection and quantitation. The LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG (NEB #L4001S) is supplied in a lyophilized format, allowing it to be shipped and stored at room temperature prior to use. The LyoPrime Luna mix can be reconstituted simply by adding water, and contains all the necessary components for One-Step RT-qPCR except primers, probe(s) and RNA sample. 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 LyoPrime Luna mix also features dUTP and thermolabile 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.

Storage

Prior to use, the LyoPrime Luna Mix should be stored at room temperature (15°C to 25°C) and protected from light. The product has a shelf life of 24 months when stored properly under these conditions. The glass vial, cap and rubber stopper are impermeable to moisture, allowing storage without desiccant. After the cap and stopper are removed, the product should be rehydrated within 1 hour (see instructions below). After rehydration, unused master mix can be stored at -20°C for up to 3 months or at 4°C for up to 3 weeks.

Rehydration

For use, remove the vial cap and stopper, add 675 μl nuclease-free water, and briefly mix by pipetting or gentle vortexing to reconstitute the lyophilized reagent cake as a 2X Master Mix. (To accommodate larger sample volumes, the lyophilized mix can instead be reconstituted as a 4X Master Mix by adding 337.5 μl nuclease free water.)

Rehydration is rapid, and the lyophilized cake will typically dissolve within 1-2 seconds after addition of water. Note that air from the cake will naturally degas from solution over ~20 seconds or after gentle vortexing.

Reaction Setup

1. Rehydrate LyoPrime Luna Mix as described above. Briefly mix other components by inversion, pipetting or gentle vortexing.
   
   If using previously hydrated material, thaw completely and then briefly mix.
   
   
2. 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 20 μl Reaction	Final Concentration
   LyoPrime Luna One-Step RT-qPCR Mix with UDG (rehydrated at 2X*)	10 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

   
   
   * If rehydrated at 4X, add 5 μl 4X Mix per 20 μl reaction.
   
   
3. Mix thoroughly but gently by pipetting or vortexing. Collect liquid to the bottom of the tube by brief centrifugation
   
   
4. Aliquot assay mix into qPCR tubes or plate. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles.
   
   
5. 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.
   
   
6. Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).
   
   

Programming the Real-Time Instrument

* 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

Primer and probe design

We recommend using primer design software (e.g., Primer3) to select target, primer, and probe sequences to maximize amplification efficiency while minimizing nonspecific amplification and primer dimers. Luna qPCR is also compatible with commercially available qPCR assays. If designing primers manually, we encourage designing short amplicons (70 bp to 200 bp) with balanced GC content (40-60%). For primers and probes, aim for a Tm of approximately 60°C using Hot Start Taq settings in the NEB Tm calculator. Where possible, it is advisable to design primers across known splicing sites (exon-exon junctions) to prevent amplification from genomic DNA.

Use in SARS-CoV-2 assays

The LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG provides sensitive detection of SARS-CoV-2 RNA, as assessed by multiplex RT-qPCR targeting SARS-CoV-2 N1 and N2 (CDC) and human RNase P (control). Additional protocol and testing information can also be found here. Primers and probes for this assay are listed below and included in the Luna^® SARS-CoV-2 RT-qPCR Multiplex Assay Kit (NEB #E3019).

SARS-CoV-2 RT-qPCR Primer/Probe Sequences (5′→ 3′)

2019-nCoV_N1 Primer/Probe Set Sequences
2019-nCoV_N1 Forward Primer:  5′ GAC CCC AAA ATC AGC GAA AT 3′
2019-nCoV_N1 Reverse Primer:  5′ TCT GGT TAC TGC CAG TTG AAT CTG 3′
2019-nCoV_N1 Probe:  5′ HEX-ACC CCG CAT TAC GTT TGG TGG ACC-Q 3′

2019-nCoV_N2 Primer/Probe Set Sequences
2019-nCoV_N2 Forward Primer:  5′ TTA CAA ACA TTG GCC GCA AA 3′
2019-nCoV_N2 Reverse Primer:  5′ GCG CGA CAT TCC GAA GAA 3′
2019-nCoV_N2 Probe:  5′ 6-FAM-ACA ATT TGC CCC CAG CGC TTC AG-Q 3′

RNase P Primer/Probe Set Sequences
RNase P Forward Primer:  5′ AGA TTT GGA CCT GCG AGC G 3′
RNase P Reverse Primer:  5′ CAA CTG AAT AGC CAA GGT GAG C 3′
RNase P Probe:  5′ Cy5-TTC TGA CCT GAA GGC TCT GCG CG-Q 3′

Note: Q=quencher

A 10X Primer/Probe Mix for this assay can be prepared as follows:

Please see the table below for an example reaction setup with the above SARS-CoV-2 Primer/Probe Mix.

* If rehydrated at 4X, add 5 μl 4X Mix per 20 μl reaction.
** Final concentrations for primers are 0.4 μM each and probes are 0.2 μM each.