The following factors are of critical importance:
• Transfection efficiency - At least 80% of the cell population should be transfected, in order to generate meaningful data. For most cell types including adherent primary cells and many suspension cell lines HiPerFect Transfction Reagent allows high transfction efficiency in combination with minimal cytotoxicity. If a high transfction efficiency is unattainable in the initial transfection, it may be necessary to use expression vectors that enable you to enrich for transfected cells by fluorescently activated cell sorting (FACS), or to select for stably transfected cells by antibiotic selection such as SureSilencing shPlasmids.
• Original abundance of the transcript - qRT-PCR reproducibility is greater at lower threshold cycle values (or higher levels of expression) than at higher threshold cycle values (or lower levels of expression). A high level of qRT-PCR reproducibility is necessary to reliably validate gene expression knock down by RNA interference.
• Biological replicate reproducibility - This includes the reproducibility of any step involving manipulation of the cell line under study (transfection, cell sorting, antibiotic selection, cell culturing, RNA isolation, protein isolation, etc.).
• Technical assay reproducibility - This includes the reproducibility of the validation and functional assays (qRT-PCR, ELISA, flow cytometry, enzymatic activity assay, etc.).