Tips from the Bench: Transfection Tips

Forward and Reverse Transfection Protocols

Forward Transfection

The most routinely employed transfection protocol where cells are seeded a day prior to transfection is referred to as “forward transfection”. Forward transfection methods work well for most adherent cell types that are seeded a day prior to transfection in order to achieve an actively dividing cell population at the time of transfection. A typical forward transfection protocol using TransIT®-2020 Transfection Reagent can be found here.

Reverse Transfection

For suspension cells and/or high throughput applications, a “reverse transfection” protocol where freshly passaged cells are added to pre-plated transfection complexes is ideal as it reduces hands-on time for the end user. Cell culture time can be further reduced by using frozen assay ready cells for some experiments. Reverse transfections are also compatible with most automated robotic systems.

The key difference between forward and reverse transfections is the cell density at the time of plating. Cells lose a day of doubling in a typical reverse transfection protocol; therefore, approximately twice the number of cells are recommended compared to a forward transfection. Typical cell densities for reverse transfection fall in the range of 3.2-4 x 105 cells per ml of complete growth media. Additionally, since reverse transfections are typically carried out in high throughout format, e.g., 96-well, 384-well, 1536-well microplates, etc., very low volumes of the transfection reagent, DNA as well as the cells are required per well. When permissible, larger transfection master mixes should be prepared with 20% additional volume to account for pipetting and dispensing errors. All of the TransIT® transfection reagents can be used for reverse transfections and diluted using 80-100% ethanol depending upon the reagent. Alternatively, researchers can dilute the required volume of reagent and DNA ten-fold in Opti-MEM® I Reduced-Serum Medium immediately before use to avoid pipetting errors. Detailed reverse transfection protocols for plasmid DNA and siRNA can be found using the following links:

Reverse transfection protocol for plasmid DNA using TransIT®-2020 Transfection Reagent

Reverse transfection protocol for siRNA using TransIT-TKO® Transfection Reagent

Reverse Transfection Protocol WorkFlow

Typical Reverse Transfection Protocol Workflow. On the day of transfection, complexes are prepared in a multiwell plate and incubated for the recommended complex formation time. Freshly passaged cells are then directly added to the multiwell plates containing the transfection complexes at twice the density of a standard forward transfection protocol. This reduces hands-on time for the end-user by one day compared to forward transfection and makes the protocol amenable to liquid handling.

There are also a few variations of standard reverse transfection protocols that are employed by researchers; brief explanations are included below:

Modified Reverse Transfection

In “modified reverse” transfections, cells are passaged and plated immediately before transfection complexes are added to the cells. In this scenario, adherent cells are loosely adhered to the plate surface by the time they interact with the transfection complexes.

Solid Phase Reverse Transfection

If the nucleic acid to be transfected is immobilized or spotted in a multi-well format already as in the case of cDNA/shRNA/siRNA screens, the transfection protocol is referred to as “solid phase reverse transfection”.

Transfection Reagents That Can be Used for Reverse Transfection

All of the TransIT® transfection reagents can be used for reverse transfections with the following considerations based on the nucleic acid to be delivered:

  1. Plasmid DNA including cDNA and shRNA encoding plasmid DNA libraries: TransIT®-Express Transfection Reagent is a low toxicity DNA transfection reagent specifically designed for high throughout applications and works well in routinely used cell types. Newer high performance transfection reagents such as TransIT®-2020 Transfection Reagent can also be used for the reverse transfection of DNA into hard-to-transfect cells. Additionally, all of the TransIT® cell line specific transfection reagents can be used for reverse transfecting plasmid DNA into respective cell types. A detailed protocol as well as citations using TransIT® reagents for reverse transfection of plasmid DNA can be found here.
  2. siRNA/miRNA: For high throughput transfection of siRNA/miRNA, either TransIT-TKO® or TransIT-siQUEST® Transfection Reagent can be used depending on the cell type. A detailed protocol as well as citations using TransIT-TKO® reagent for the reverse transfection of siRNA can be found here.
  3. Large mRNA: Reverse transfection of larger RNA species such as mRNA can be performed using TransIT®-mRNA Transfection Kit.
  4. DNA and RNA Oligonucleotides: High throughput reverse transfection of different DNA and RNA oligonucleotides can be performed using TransIT®-Oligo Transfection Reagent.

Citations for Reverse Transfection using TransIT® Transfection Reagents

Citation Cell Type Transfected Nucleic Acid Transfected TransIT® Reagent Used Robotic system used Multi-well format Application
Zhao et al. Molecular Neurodegeneration 2009, 4:4 HeLa Plasmid DNA TransIT®-LT1 None 384-well HT fluorescence polarization-based Aβ degradation assay
Owens et al. J Biol Chem. 2010 February 26; 285(9): 6761-6769. HeLa Plasmid DNA TransIT®-LT1 Multidrop 384 (Titan) 384-well HT cell-based screens to detect stabilized protein targets following chemical mutagenesis
Warzecha et al. Mol Cell. 2009 March 13; 33(5): 591-601. HEK 293T cell clone stably expressing the luciferase splicing reporter Plasmid DNA TransIT®-293 Wellmate Handler (Matrix) 384-well HT cell-based genome-wide cDNA expression screening
Andersen et al. Mol Ther. 2010 November; 18(11): 2018-2027. Human mesenchymal stem cells (hMSCs) siRNA TransIT-TKO® None Tissue culture plates coated by a lyophilization process with TransIT-TKO®/siRNA particles Tissue engineering using siRNA coated nanostructured scaffolds
Lu et al. Oncogene. 2011 November 10; 30(45): 4567-4577. MDA-MB-468 siRNA TransIT-TKO® None 96-well HT Kinome siRNA phosphoproteomic screen using an siRNA library targeting 541 kinases and kinase-related genes
Choudhary et al. J Biol Chem, 286, 37187-37195. A549-Luc stable reporter cell line siRNA TransIT-siQUEST® Titertek, multidrop 384 cell dispenser 96-well HT siRNA screening of the human kinome
Liu et al. J Biol Chem. 2008 August 22; 283(34) 23169-23178. Ikkγ-/- MEFs siRNA TransIT-siQUEST® None Not specified Mechanism study for RSV infection

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