SiRNA knock down - HuH7 cells - 24 well plate - biochemical analysis (Thiele lab)

From LipidomicsWiki

siRNA transfection – HuH7 cell line – 24 well plate

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 HuH7 cell line can be transfected under serum conditions by using two different reagents

Transfection reagent 1: Hiperfect, Invitrogen
siRNA stock: 2 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 7 µl of siRNA stock with 100 µl Opti-MEM
•    Add 6 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.

Transfection reagent 2: Interferin, BioMol
siRNA stock: 125 nM

Step 1:
Plate the cells in 500 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 7 µl of siRNA stock with 100 µl Opti-MEM
•    Add 2 µl Hiperfect
•    Mix
•    Incubate 10 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.