Reverse Transfection Protocol for DNA

All TransIT® DNA transfection reagents, including the new TransIT-X2® Dynamic Delivery System, can be used for reverse transfection of DNA into multiple cell types. Additionally, cryopreserved cell stocks can be utilized for immediate transfection reducing overall culture time. Data showing reverse transfection of normally trypsinized and frozen spun cells can be found here.

Reverse Transfection Protocol for DNA in 96-well Plates Using
TransIT-X2® Dynamic Delivery System

An example of high efficiency forward and reverse transfection of A549 cells using the broad spectrum TransIT-X2® Dynamic Delivery System can be found in the figure below.

Comparison of Forward and Reverse Transfection in A549 Cells Using TransIT-X2 Dynamic Delivery System

Comparison of Forward and Reverse Transfection in A549 Cells Using TransIT-X2® Dynamic Delivery System. A549 cells were seeded 24 hours prior to transfection for forward transfection (A). For reverse transfection (B), A549 cells were plated after adding complexes following the protocol below. Cells were transfected with a firefly luciferase encoding plasmid DNA (0.1 µg DNA) using either TransIT-X2® Dynamic Delivery System or Lipofectamine® 2000 Transfection Reagent at the indicated reagent-to-DNA ratios in a 96-well plate for both forward and reverse transfection protocols. Luciferase activity was measured at 24 hours post-transfection using a conventional luciferase assay. Both forward and reverse transfection using TransIT-X2® Dynamic Delivery System yield superior luciferase expression compared to Lipofectamine® 2000.

Before You Start

Akin to forward transfection, optimizing transfection conditions by transfecting a reporter plasmid into the cell type of interest (such as a luciferase or GFP encoding plasmid) into cells is critical prior to high throughout screening.

  • For best results, determine the appropriate dose of DNA and transfection reagent in the multi-well format for the screen. Specific tips on optimizing plasmid DNA transfection can be found here. For example, the optimal DNA concentration generally ranges between 0.05-0.2 μg/well of a 96-well plate with an optimal TransIT-X2®:DNA ratio of 2-6 μl per 1 μg DNA.
  • Observe reporter gene expression and toxicity at regular time points over at least a 24 hour period, optimally for 48 hours.

Use the optimal DNA and transfection reagent dosage for HT screening applications.

A. Cell Plating Prior to Transfection

  1. At least 24 hours prior to transfection, plate cells at an appropriate cell density in a T-75 cm2 flask or similar tissue culture dish so that the cells will be 70-80% confluent the following day. Approximately 2-6 x 106 cells will be needed per 96-well plate. Multiple flasks may need to be prepared if more than one 96-well plate is to be transfected.
  2. Incubate the cells overnight.

B. Complex Formation for 96-well Plates

  1. In each well of the 96-well plate to be used for transfection, add appropriate amount of serum-free medium (i.e. Opti-MEM® I Reduced Serum Medium) (see Table 1). Note: Alternatively, a mastermix can be prepared in a sterile tube if transfecting the same plasmid throughout the plate. Calculations are shown in Table 1 for 120 wells to account for pipetting errors. If a transfection master mix is prepared, prepare the transfection mixture as follows (Steps 1-4) and add per well to the 96-well plate using a multi-channel pipettor or liquid handler after complex formation (Step 4).
  2. Add appropriate amount of stock plasmid DNA (see Table 1) to the wells containing the each well of the 96-well plate.
  3. Add appropriate amount of TransIT-X2® to the Opti-MEM® I plasmid DNA mixture (see Table 1).
  4. Incubate at room temperature for at least 15 minutes to allow for the transfection complexes to form. Note: If you are using pre-printed cDNA/shRNA screens, allow for an additional 10 minutes for the reconstitution of the dried printed DNA. Do not allow complexes to incubate longer than 60 minutes before adding cells from step C below.

C. Cell Plating in 96-well Plates

  1. Trypsinize cells in T-75 cm2 flask (from Step A) as per standard tissue culture procedure. Note: To prevent re-adherence of the cells to the flask, perform this step no more than one hour prior to transfection. To further reduce cell culture time, cell plating can also be performed using cryopreserved cell stocks, after they are centrifuged to remove DMSO and counted using Trypan Blue.
  2. Add 5-10 ml of complete media to the cell suspension. Mix thoroughly by pipetting.
  3. Count the cells using a hemacytometer to determine the appropriate volume of cells in media to obtain 1.6-4.8 x 105 cells per ml.
  4. Add 92 µl of diluted cell mixture (1.4-4.4 x 104 cells) to each well. Gently rock the dish back and forth and from side to side to distribute the cells evenly. Do not swirl or rotate the dish, as this may result in uneven distribution.
  5. Incubate 24-48 hours.
  6. Harvest and assay for gene expression or other reporter assay.

Table 1. Recommended starting conditions for reverse DNA transfections with TransIT-X2® System

  Volume needed per well of a 96-well plate Total volume needed if preparing a mastermix for a 96-well plate*
Volume of serum free media for transfection complex formation 9 µl 9 x 120 = 1080 µl =1.08 mls
Amount of DNA needed per well ‡ 0.1 µg 0.1 x 120 = 12 µg
Volume of TransIT-X2® per well ‡ 0.3 µl 0.3 x 120 = 36 µl
Trypsinized cells in complete growth medium 92 µl 92 x 120 = 11,040 ul = 11.04 mls

* The mastermix calculations are based on 120 wells to account for pipetting errors.

‡ If small volumes of TransIT-X2® and plasmid DNA need to be pipetted, dilute the required volume of reagent and DNA ten-fold in Opti-MEM® I Reduced-Serum Medium before each use to avoid pipetting errors. Do not store diluted TransIT-X2® or DNA stocks.

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