High Throughput RNAi Screening Protocol

Increased availability of synthetic siRNA screens in the recent years has positioned multiwell RNAi screening as a powerful technique for understanding gene function. Delivery of smaller nucleic acids such as siRNA and miRNA is relatively straightforward as delivery to the cytoplasm is sufficient to initiate gene knockdown. Mirus offers three different reagents for siRNA or miRNA transfection - TransIT-X2® Dynamic Delivery System, TransIT-TKO® and TransIT-siQUEST®. Any of these three reagents can be used for reverse transfecting short species of RNA such as siRNA and miRNA into a broad range of cell types. TransIT-X2® Dynamic Delivery System exhibits high knockdown with either forward or reverse transfection; see data below followed by a detailed reverse transfection protocol using TransIT-X2® Dynamic Delivery System in a 96-well format.

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

High Knockdown Using TransIT-X2 System for Forward or Reverse Transfection

High Knockdown Using TransIT-X2® System for Forward or Reverse Transfection in CHO Cells Stably Expressing Firefly Luciferase. For the forward transfection (blue), CHO-luc cells were seeded in 96-well plates 24 hours prior to transfection; whereas, for reverse transfection (red), cells were plated after adding complexes following the protocol below.  Cells were transfected with 25 nM of either a non-targeting siRNA or a anti-firefly luciferase siRNA in complex with different amounts of TransIT-X2® Dynamic Delivery System (indicated beneath each bar). Luciferase expression, normalized to non-targeting siRNA control were measured at 24 hours post-transfection. Using either forward or reverse transfection,  high knockdown is  observed with a broad range of TransIT-X2®:siRNA ratios.

Before You Start

Optimizing knockdown conditions in the cell type of interest by transfecting a RNAi reporter (such as a luciferase targeting siRNA) before conducting an HT RNAi screen is critical, if allowed by the experimental set-up. Alternatively, siRNAs targeting a non-essential endogenous gene can be employed. There are also options to directly track siRNA delivery by using labeled RNAi controls such as Label IT® RNAi Delivery Controls. Specific tips on optimizing siRNA transfection can be found here. A comprehensive resource on additional considerations for high throughout RNAi screening such as siRNA specificity and controlling off-target effects can be found here (1).

  • Determine the appropriate dose of siRNA (10-50 nM final concentration per well) and TransIT-X2® (0.2-0.6 µl) per well of the 96-well format to be used for HT RNAi screening.
  • Observe reporter knockdown and toxicity at regular time points over at least a 48 hour period, optimally for 72 hours.

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 siRNA 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 tansfection 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 siRNA (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 siRNA 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 preprinted siRNA screens, allow for an additional 10 minutes for the reconstitution of the dried printed siRNA. Do not allow complexes to incubate longer than 60 minutes before adding cells as per 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 knockdown of gene expression or other reporter assay.

Table 1. Recommended starting conditions for reverse siRNA 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 ml
Amount of siRNA needed per well (1 µM stock) (25 nM final) ‡ 2.5 µl 2.5 x 120 = 300 µl
Volume of TransIT-X2® per well ‡ 0.3 µl 0.3 x 120 = 40 µl
Trypsinized cells in complete growth medium 92 µl 92 x 120 = 11,040 ul = 11.04 ml

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

‡ If small volumes of TransIT-X2® and siRNA 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 siRNA stocks.

Reference:

1. High-throughput RNAi screening in cultured cells: a user's guide. Christophe J. Echeverri & Norbert Perrimon. Nature Reviews Genetics 7, 373-384 (May 2006)

High throughput shRNA library screening

While most routinely used cell lines such as HEK 293, CHO-K1, etc. take up siRNA efficiently using transfection reagents, hard-to-transfect cell types such as immune derived cells can be quite refractory to such methods. An alternative method for RNAi screening in these cells is via recombinant lentiviruses that integrate into the cellular genome and encode an shRNA that can be converted into an siRNA inside the cell. Use of shRNA encoding RNAi libraries also offers additional advantages such as:

  • Highly parallel processing of RNAi screens in a pooled format compared to synthetic siRNA screens
  • Longer lasting gene knockdown that might be required to analyze certain long term cellular functions as the integrated virus continues to express shRNA through multiple cell division cycles.
  • Antibiotic selection via the lentiviral backbone to allow efficient selection of transduced cells encoding the shRNA
  • Inducible screening in case of shRNA libraries where the shRNA is encoded downstream of an tetracycline inducible promoter, allowing scope for more experimental manipulation compared to constitutive expression.

A very comprehensive resource for generation of lentiviral shRNA libraries can be found at the public protocol database of the RNAi Consortium (Broad Institute of MIT and Harvard). Recombinant lentiviral particles can be easily generated after transfection of producer cell types such as HEK293T using Mirus broad spectrum DNA transfection reagents. Further information on using Mirus' transfection reagents for virus production can be found here.


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