Transfection efficiency control DsiRNAs
Good transfection is required for successful RNA interference using DsiRNAs. We recommend optimizing transfection conditions for each cell line studied and for each form of nucleic acid used (for example, large DNA plasmids often require different transfection conditions than shorter DsiRNAs). It may also be necessary to empirically test different transfection reagents (or other approaches) to establish a protocol that performs optimally with each cell line used.
The dye-labeled transfection efficiency control DsiRNAs allow for rapid, easy screening of many reagents or conditions in parallel.
Endogenous positive control DsiRNAs and qPCR assays
It is possible to get good DsiRNA uptake without delivery of the oligos into the correct cytoplasmic location for effective RNAi. We recommend testing for functional knockdown using a positive control DsiRNA after checking for efficient transfection.
The HPRT-S1 Positive Control DsiRNA can be used for this purpose. With good transfection, 10 nM HPRT positive control will reduce HPRT mRNA levels by >90% at 24 hours. These controls are intended only for developing good transfection methods and knockdown is best examined at 24 or 48 hour time points. Knockdown of HPRT can slow cell growth and affect cell viability for incubation periods >72 hours. Due to sequence similarity, the HPRT-S1 control DsiRNA can be used in human, mouse, rat, and Chinese hamster (CHO) cells. Other genomes may require custom controls.
HPRT qPCR assays are available for measuring knockdown of HPRT mRNA expression in human and mouse cells.
Exogenous reporter gene control DsiRNAs
Depending on your cells, knockdown of reporter genes can be used as either positive or negative controls. For cell lines that express the EGFP or Luciferase (Firefly or Renilla) reporter gene stably or by co-transfection of an expression plasmid, DsiRNAs targeting the respective reporter gene serve as positive controls. However, for cell lines that do not express EGFP or Luciferase reporter genes, DsiRNAs targeting the respective reporter gene can serve as negative controls. These reporter gene controls are validated, functional DsiRNAs with efficient RISC loading and, therefore, offer more control than non-targeting sequences.
Universal negative control DsiRNAs
The Negative Control DsiRNA is non-targeting DsiRNA that will not interact with any sequences in the human, mouse, or rat transcriptomes. If making a choice, we recommend using the Negative Control DsiRNA, instead of the Scrambled Negative Control DsiRNA.
For cells that do not express the respective reporter gene, the EGFP and luciferase DsiRNAs may be used as negative controls if functional, targeting DsiRNA are desired (see Exogenous reporter gene positive controls above).