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In a six well tissue culture plate, seed 2 x 105 cells per well in 2 ml antibiotic-free normal growth medium supplemented with FBS. |
| NOTE: |
This protocol is recommended for a well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes. |
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Incubate the cells at 37° C in a CO2 incubator until the cells are 60 - 80% confluent. This will usually take 18-24 hours. |
| NOTE: |
Healthy and subconfluent cells are required for successful transfection experiments. It is recommended to ensure cell viability one day prior to transfection. |
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Prepare the following solutions: |
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Solution A: For each transfection, dilute 2-8 µl of siRNA duplex (i.e., 0.25-1 µg or 20-80 pmols siRNA) into 100 µl siRNA Transfection Medium: sc-36868. |
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Solution B: For each transfection, dilute 2-8 µl of siRNA Transfection Reagent: sc-29528 into 100 µl siRNA Transfection Medium: sc-36868. Peak activity should be at about 6 µl siRNA Transfection Reagent. |
| NOTE: |
Do not add serum and antibiotics to the siRNA Transfection Medium: sc-36868 |
| NOTE: |
Optimal siRNA amount used for transfection may vary for each target protein and should be determined experimentally. |
| NOTE: |
If a lower siRNA concentration is desired, dilute siRNA appropriately with siRNA Dilution Buffer: sc-29527. |
| NOTE: |
Although highly efficient in a variety of cell lines, siRNA Transfection Reagent: sc-29528 may not be suitable for use with all cell lines. |
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Add the siRNA duplex solution (Solution A) directly to the dilute Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15–45 minutes at room temperature. |
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Wash the cells once with 2 ml of siRNA Transfection Medium: sc-36868 Aspirate the medium and proceed immediately to the next step. |
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For each transfection, add 0.8 ml siRNA Transfection Medium to each tube containing the siRNA Transfection Reagent mixture (Solution A + Solution B). MIx gently and overlay the mixture onto the washed cells |
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Incubate the cells 5–7 hours at 37° C in a CO2 incubator. |
| NOTE: |
Longer transfection times may be desirable depending on the cell line. However prolonged serum starvation may result in unwanted cell detachment or death. |
| NOTE: |
Fluorescein Conjugated Control siRNA should only be incubated for a total 5-7 hours at 37° C in a CO2 incubator. At the end of incubation they are ready to be assayed by fluorescent microscopy. |
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Add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium) without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with 1x normal growth medium. |
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Incubate the cells for an additional 18–24 hours. |
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Aspirate the medium and replace with fresh 1x normal growth medium. |
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Assay the cells using the appropriate protocol 24–72 hours after the addition of fresh medium in the step above. |
| NOTE: |
Controls should always be included in siRNA experiments. Use either Control siRNAs: sc-37007, sc-44230,sc-44231, sc-44232, sc-44233, sc-44234, sc-44235, sc-44236, sc-44237 or sc-44238 or Control siRNA (Fluorescein Conjugates): sc-36869, sc-44239, sc-44240 or sc-44241. Each contain a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA. |
| NOTE: |
For Western blot analysis prepare cell lysate as follows: Wash cells once with PBS. Lyse cells in 300 µl 1x electrophoresis sample Buffer (sc-24945: Electrophoresis Sample Buffer, 2X) by gently rocking the 6 well plate or by pipetting up and down. Sonicate the lysate on ice if necessary. |
| NOTE: |
For RT-PCR analysis isolate RNA using the method described by Chomczynski and Sacchi (Anal Biochem. 1987 Apr;162(1):156-159. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Chomczynski P, Sacchi N.) or a commercially available RNA isolation kit. |
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siRNA Mediated Inhibition of Gene Expression |
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