Protocol for Luna^® Cell Ready Probe One-Step RT-qPCR Kit (NEB #E3031)

Before Use

• Prepare cells in advance (various cell types such as adherent, suspension, or cryopreserved can be used) and ensure cells are intact before lysis.

• Consider testing several cell dilutions to determine the appropriate input range to use for lysis and RT-qPCR. Although a standard curve is not required for high throughput screening experiments, it is still recommended.

• It is important to use only a monolayer of cells in well-plate formats. We recommend using Surface Areas and Guide for Recommended Medium Volumes from Corning^® as a starting point for preparing cell cultures.

• Ensure that all components are thawed and mixed prior to use. Once thawed, place on ice prior to use. Workflow using Luna Cell
  Ready One-Step RT-qPCR Kit.

Workflow using Luna Cell Ready One-Step RT-qPCR Kit

Part I. Cell Lysate Preparation using Luna Cell Ready Lysis Module

Step 1. Processing Cells

Cell Resuspension Solution (CRS) is recommended for resuspending/diluting cells to reduce RNA damage during the handling process. When only a fraction of the whole sample is to be used (e.g., adherent cells grown in vessels other than 24/48/96/384 well plates, suspension cells, or cryopreserved cells), you should resuspend with CRS to protect your samples’ RNA during processing. CRS is not required for adherent cells in 24/48/96/384 well plates.

Prepare Cell Resuspension Solution (CRS) by diluting Luna Cell Ready RNA Protection Reagent (25X) to 1X with cold PBS (e.g. for each sample, mix 2 μl Luna Cell Ready RNA Protection Reagent with 48 μl of 1X PBS to make 50 μl Cell Resuspension Solution).

* For high throughput screening (e.g., adherent cells in 96 wells or 384 well plates), cell wash is optional if medium removal can be completed via a plate flipping step.

Step 2. Prepare Cell Lysis Mix

1. Thaw Luna Cell Ready Lysis Buffer and Luna Cell Ready Stop Solution at room temperature, then place on ice with all other components. After thawing completely, briefly mix each component by inversion.

2. Prepare Cell Lysis Mix of all components, adding the Luna Cell Ready Protease immediately before use. Mix thoroughly by pipetting gently. Centrifuge briefly to collect the solution to the bottom of the tube and store on ice.
   For best results, the Cell Lysis Mix should be used immediately (within 15 minutes).
   
   

   COMPONENT	40 µl CELL LYSIS MIX	FINAL CONCENTRATION
   Luna Cell Ready Lysis Buffer (2X)	25 μl	1X
   DNase I (RNase-free) (10X)	5 μl	1X
   Luna Cell Ready RNA Protection Reagent (25X)	2 μl	1X
   Luna Cell Ready Protease (25X)	2 μl	1X
   Nuclease-free Water	6 μl

   Up to 2,000 cells per μl lysis reaction is recommended. In a typical 50 μl lysis reaction, 100,000 cells can be lysed.

Step 3. Cell Lysis

For lysis using cells resuspended from adherent cells grown in vessels other than 24/48/96/384 well plates, suspension cells, or cryopreserved cells: Mix up to 5 μl of cells resuspended in CRS with the Cell Lysis Mix to a final volume of 45 μl. Gently pipet up and down 6 times. Incubate the lysis reaction at 37°C for 10 min.*

For lysis of adherent cells in 24/48/96/384 well plates: Aliquot an appropriate volume of Cell Lysis Mix into each well as indicated in the following table and incubate the reaction at 37°C for 10 min.*

For most efficient lysis, automatic shaking is recommended for cell densities higher than 200 cells/μl.

*Note: Lysis at room temperature is an option if cell density is less than 200 cells/μl in the lysate.

Step 4. Lysis Termination

Add 10X Luna Cell Ready Stop Solution to a final concentration of 1X and mix well by pipetting up and down 6 times. Centrifuge briefly to collect the solution to the bottom of the tube. Incubate at 25°C for 5 min.

For 96 well plates, we found that adding 5 μl of Luna Cell Ready Stop Solution (10X) to the 40 μl lysis solution (± 10% cells and residual wash buffer) was sufficient for complete lysis.

Cell lysates can be stored on ice for up to five hours, at -20°C for up to five days and at -80°C for long term storage. Cell lysate is stable for up to five freeze and thaw procedures.

Part II. RNA Detection using using Luna Universal Probe One-Step RT-qPCR Kit 

For best results, it is recommended to run each lysate sample and control in triplicate.

1. Determine the total volume for the appropriate number of reactions (adding 10% overage) and prepare an assay mix of all components except for cell lysate. Mix thoroughly by gentle pipetting or vortexing. Centrifuge briefly to collect the solution to the bottom of the tube.

   COMPONENT	20 μl REACTION	FINAL CONCENTRATION
   Luna Universal Probe One-Step Reaction Mix (2X)	10 μl	1X
   Luna WarmStart RT Enzyme Mix (20X)	1 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
   Cell Lysate	variable	≤ 2 μl* (up to 4,000 cells)
   Nuclease-free Water	to 20 μl

   *In general, 1 μl of cell lysate is recommended. However, lysate input may be optimized for best results. It is not recommended for the lysate to exceed more than 10% of the RT-qPCR reaction volume.
   
   

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

3. Add cell lysate 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). RT-qPCR experiment should proceed immediately after setup for best results.
   
   

5. Program real-time instrument with indicated thermocycling protocol (see table below). Ensure that a plate read is included at the end of the extension step.
   
   

6. Confirm selection of appropriate detection channel(s) for the fluorophore(s) present in the assay in the real-time instrument.
   
   

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

   CYCLE STEP	TEMP	TIME	CYCLES
   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)	40–45

   *A 55°C RT step temperature is optimal for Luna WarmStart Reverse Transcriptase. To insure best performance and full WarmStart activation avoid using a temperature of < 50oC.

   ** For Applied Biosystems real-time instruments use a 60 second extension step.