SiRNA knock down - HeLa cells - 24 well plate - biochemical analysis (Thiele lab)

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siRNA transfection – HELA cell line – 24 well plate – biochemical analysis

This reverse genetic approach is used to knock down specific genes followed by biochemical analysis (e.g. western blotting for specific proteins, lipid analysis by mass spectrometry, metabolic analysis using radioactive lipid precursors) of the resulting phenotype. 

The HELA cell line can be transfected under serum conditions and under serum-free conditions.

siRNA transfection under serum conditions:
Transfection reagent: Hiperfect, Invitrogen
siRNA stock: 5 µM

Step 1:
Plate the cells in 400 µl complete culture medium at least one day before the transfection in a 24 well plate.

Step 2:
Day of transfection: cells should be 15 % confluent.
Preparation of the transfection solution per well:
•    Mix 2 µl of siRNA stock with 100 µl Opti-MEM
•    Add 3 µl Hiperfect
•    Mix
•    Incubate 5 min
•    Add 100 µl of the transfection solution to the well
•    Total volume in the well: 500 µl

Step 3:
Incubate the plate in a cell culture incubator for 24-96 hours. The incubation time depends on the target protein and the question under study.

Step 4:
Check knock-down efficiency on mRNA- or protein-level by qRT-PCR or Western-Blotting, respectively. Then proceed with further analysis.

siRNA transfection under serum-free conditions:
Transfection reagent: Oligofectamine, Invitrogen
siRNA stock: 5 µM

Step 1:
Plate the cells in complete culture medium at least one day before the transfection in a 24 well plate.

Step 2:
Day of transfection: cells should be 15 -20 % confluent.
-    remove culture medium, add 250 µl OPTIMEM per well
Preparation of transfection solution per well:
-    1) 10 µl siRNA plus 75 µl OPTIMEM
-    2) 2 µl Oligofectamine plus 13 µl OPTIMEM
-    mix each solution
-    incubate 5 min
-    add solution 1 to solution 2
-    mix
-    incubate 20min
-    add 100 µl of transfection solution to the well
After 4 hours add 500µl complete culture medium per well.

Step 3:
Incubate the plate in a cell culture incubator for 24-96 hours. The incubation time depends on the target protein and the question under study.

Step 4:
Check knock-down efficiency on mRNA- or protein-level by qRT-PCR or Western-Blotting, respectively. Then proceed with further analysis.