Oligofectamine™ Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Do not add antibiotics to media during transfection; this causes cell death.	Mix all the complex reactions and incubate the cells at 37°C in a CO2 incubator for 4 hours. Add 50 µL of growth medium containing 3X the normal concentration of serum without removing the transfection mixture and assay for gene activity at 24–72 hours post-transfection.

Upstream tips
Do not add antibiotics to media during transfection; this causes cell death.
Protocol tips
Mix all the complex reactions and incubate the cells at 37°C in a CO2 incubator for 4 hours. Add 50 µL of growth medium containing 3X the normal concentration of serum without removing the transfection mixture and assay for gene activity at 24–72 hours post-transfection.

Publication protocol

Small interfering RNA (siRNA) against GRK4 mRNA and its control scrambled RNA were synthesized and purified with reverse-phase high-performance liquid chromatography as 25-mer phosphorothioate-modified oligodeoxynucleotides (GRK4 siRNA sequence: #1 5’-AUCUAAAGAGGUGCAUUGAAUUCUUdTdT-3’, #2 5’-AAGGACCUCAAUGAAUAUGAAGAUAdTdT-3’; scrambled RNA sequence: 5′-TGACGATAAGAACAATAACdTdT-3′), from nucleotides 412 to 436 and 1752 to 1776 of the rat GRK4 cDNA.

The effects of 50 nM siRNA were compared with scrambled RNA (control). Briefly, cells were grown in 6-well plates until 60% confluence, and 50 nM siRNA or control RNA were mixed with 6 μL of oligofectamine in Optimem medium (Invitrogen Life Technologies) and incubated for 24 hr, then switched to growth medium and incubated for another 24 hr. The cells were collected and processed for RT-PCR for GRK4 to determine the efficiency of siRNA-induced GRK4 gene silencing (Supplementary Figure S3).

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Manufacturer protocol

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