Protocol for Luna® Cell Ready Lysis Module (NEB #E3032)

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.

  • 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.



 

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

Step 1. Processing Cells

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). Cell Resuspension Solution is recommended for resuspending/diluting cells to reduce RNA damage during the handling process.

CELL CULTURE PROCEDURE
Adherent cells in 24/48/96/384 well plates
  1. Remove all cell-culture medium.
  2. Rinse briefly with cold PBS and aspirate PBS*
Adherent cells grow in other vessels Detach cells using common sub-culturing technique
  1. Transfer the desirable volume of cells and spin down the cell pellets
  2. Rinse briefly with cold 1X PBS and aspirate PBS*
  3. Resuspend cells with CRS (up to 20,000 cells/µl). Store on ice and proceed to lysis within 10 mins
Suspension cells    
Cryopreserved cells Quickly thaw the cell stock at 37°C

 

* 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).

  3. COMPONENT

    40 μl REACTION

    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 cell resuspension: mix up to 5 μl of cells 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 in culture 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.

CULTURE PLATE

CELL LYSIS MIX

24 well

160 μl

48 well

80 μl

96 well

40 μl

384 well

8 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 5 μl of Luna Cell Ready Stop Solution (10X) to the lysis reaction (45 μl) 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.

Cell lysates can be stored on ice 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 Luna Universal One-Step RT-qPCR Kit or Probe RT-qPCR

The cell lysate can easily comprise up to 10% of the one-step RT-qPCR volume (i.e. 2 µl cell lysate in 20 µl RT-qPCR experiment). Please refer to NEB #E3030 or NEB #3031 manual Part II for details.