IDT
IDT
Validated Control DsiRNA Duplexes and other Control ReagentsFluorescent Transfection Efficiency Control DuplexesA successful RNAi experiment starts with good transfection. It is good practice to optimize transfection conditions for each cell line studied as well as for each form of nucleic acid employed (for example, large DNA plasmids often require different transfection conditions than short dsRNA oligos). It may be necessary to empirically test a number of different cationic lipids (or other approaches) to establish a protocol that performs optimally with each cell line employed. Use of a dye-labeled transfection control oligo allows for rapid, easy screening of many reagents in parallel.
When optimizing transfection methods, IDT recommends using dye-labeled oligos at 10 nM (or less); higher concentrations can increase the amount of non-specific binding which can cause background and falsely elevate the apparent success of transfection.
The following dye-labeled RNA duplexes are available as ReadyMade™ products for this purpose. RNA oligos can be labeled with other dyes as a custom order.
Endogenous Gene Positive Control Duplexes and PrimersUse of dye-labeled control oligos is not sufficient by itself to optimize transfection. It is possible to get seemingly good dye-oligo uptake without delivery of the oligos into the correct cytoplasmic location for functional RNAi. Transfection conditions that "pass" the dye-labeled study should also be tested for functional knockdown using a positive control siRNA.
The "HPRT-S1 DS Positive Control" duplex can be used for this purpose. When transfection is good, theHPRT mRNA will reduce HPRT mRNA levels by >90% at 24 hours when used at 10 nM. Please note that the HPRT control is intended only to develop good transfection methods and 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 duplex can be employed in human, mouse, rat, and Chinese hamster (CHO) cells. Other genomes may require customized controls.
IDT recommends studying functional transfection efficiency by examining mRNA levels 24 hours after transfection. Alternatively, western blots can be performed at 48–72 hours.
It is often useful to have cloned gene fragments available to establish quantitative standard curves when performing qRT-PCR. Relative RNA levels can be measured without use of copy number standards (ΔΔCt method); however, cloned copy number standards permit true quantitative measurements to be made. The assay amplicon for each of the above HPRT positive control qRT-PCR reactions has been cloned into pBlueScript™-II and sequence verified. Clones are provided as 0.5 µg of purified, linearized plasmid DNA that is directly ready for use in PCR.
A map of the plasmid clone is shipped with the control.
Internal Control Primers for qRT-PCR AnalysisWhen qRT-PCR is performed it is necessary to have an internal standard to control for RNA loading. While many different housekeeping genes have been used for this purpose (such as β-Actin, GAPDH, or Cyclophilin), most of these genes show fluctuation in expression levels with different treatments and are not as invariant as is needed for a true internal qRT-PCR control. IDT has developed the following SYBR®-Green assays suitable for use an internal normalization standard in qRT-PCR analysis (ΔΔCt method). When combined with use of the cloned copy number control plasmids, these reagents permit relative RNA mass-loading normalization plus absolute quantitative PCR to be performed.
It is often useful to have cloned gene fragments available to establish quantitative standard curves when performing qRT-PCR. Relative RNA levels can be measured without use of copy number standards (ΔΔCt method); however, cloned copy number standards permit true quantitative measurements to be made. The assay amplicon for each of the above internal control qRT-PCR reactions has been cloned into pBlueScript™-II and sequence verified. Clones are provided as 0.5 µg of purified, linearized plasmid DNA that is directly ready for use in PCR.
A map of the plasmid clone is shipped with the control.
Exogenous Reporter Gene Positive ControlsReporter genes can be used both as positive controls and as negative controls and so are very useful reagents. If your cell line expresses the reporter either stably or via co-transfection of an expression plasmid, the anti-EGFP or anti-Luciferase (Firefly or Renilla) DsiRNAs can function as a positive control. If your cell line does not express these reporter genes, then the anti-EGFP or anti-FLuc DsiRNAs can function as negative controls. Importantly, these DsiRNAs are validated, functional duplexes with known efficient RISC loading and therefore offer an added level of control that non-targeting sequences cannot offer.
Universal Negative ControlNegative control duplexes have been developed which do not target any sequence in the human, mouse, or rat transcriptomes. They can be employed as a universal negative controls for DsiRNA transfections. These are non-targeting sequences. IDT recommends the NCI negative control over the DS scrambled negative. The EGFP and FLuc DsiRNAs may also be used as negative controls if functional, targeting duplexes are desired (see Exogenous Reporter Gene Positive Controls above).
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