Figure 2.

Transfection scheme. (A) Reagents used include psiCHECK-2 reporter vector (Promega) that constitutively expresses high levels of Renilla luciferase (R_luc) and firefly luciferase (ff_luc) as well as the ampicillin resistance gene (AmpR) for propagation in bacteria. Each pair of students also tested three siRNAs: siRNA that had been previously validated to decrease expression of R_luc, a nontargeting siRNA that has no sequence homology to any human gene, and their experimental siRNA directed against a portion of the R_luc gene. (B) Pattern for student's transfection reactions. HeLa cells grown in two six-well dishes were transfected with Lipofectamine 2K (Invitrogen, Carlsbad, CA) and, from left to right, no DNA, reporter plasmid alone, validated siRNA alone, reporter plasmid plus a validated siRNA to knockdown expression of R_luc, reporter plasmid plus a nontargeting siRNA, and finally reporter plasmid plus the student-designed siRNA. Cells were fed by the teaching staff after 24 h, and students returned after 48 h to measure luciferase activity for each sample and isolate RNA.