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Validated Control DsiRNA Duplexes and other Control Reagents

The following collection of validated control reagents are available premade for immediate delivery. Inquire for custom controls for special needs.

Fluorescent Transfection Efficiency Control Duplexes

A 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. Other dyes can be provided on RNA oligos on a custom basis. For example, the LI-COR IRdye® 800 CW can be placed on RNA duplexes and employed for in vivo biofluorescence imaging, with excellent tissue penetration due to the far infrared spectral emission of this fluorophore.

Product1nm5nm
TYE 563TM DS Transfection Control$78.00 USD$195.00 USD
TEX 613TM DS Transfection Control$78.00 USD$195.00 USD
Cy3TM DS Transfection Control$94.00 USD$234.00 USD

 

Endogenous Gene Positive Control Duplexes and Primers

Use 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 if incubation extends > 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.

Product1nm5nm
HPRT-S1 DS Positive Control (Human/Mouse/Rat)$60.00 USD$150.00 USD
HPRT-Bt DS Positive Control (Cow)$60.00 USD$150.00 USD
HPRT-Bt DS Positive Control (Pig)$60.00 USD$150.00 USD


IDT recommends studying functional transfection efficiency by examining mRNA levels at 24 hours post transfection. Alternatively, Western blots can be performed at 48-72 hours. Validated primers for SYBR Green qRT-PCR assays are available for the following organisms:

SYBR-Green Q-PCR Primer Sets1nm5nm
HPRT SYBR-Green primers (Human)$25.00 USD$60.00 USD
HPRT SYBR-Green primers (Mouse)$25.00 USD$60.00 USD
HPRT SYBR-Green primers (Rat)$25.00 USD$60.00 USD
HPRT SYBR-Green primers (Chinese hamster)$25.00 USD$60.00 USD
HPRT SYBR-Green primers (Cow)$25.00 USD$60.00 USD
HPRT SYBR-Green primers (Pig)$25.00 USD$60.00 USD


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 ug of purified, linearized plasmid DNA that is directly ready for use in PCR.

Cloned Purified PlasmidsAccession #0.5 μg
0.5 µg Human HPRT Cloned qControlNM_000194$195.00 USD
0.5 µg Mouse HPRT Cloned qControlNM_013556$195.00 USD
0.5 µg Rat HPRT Cloned qControlNM_012583$195.00 USD
0.5 µg Ch Hamster HPRT Cloned qControlJ00060$195.00 USD
0.5 µg Cow HPRT Cloned qControlNM_001034035$195.00 USD


A map of the plasmid clone is shipped with the control.

Internal Control Primers for qRT-PCR Analysis

When 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.

Product1nm5nm
RPLP0 SYBR-Green primers (Human)$25.00 USD$60.00 USD
RPL23 SYBR-Green primers (Mouse)$25.00 USD$60.00 USD
RPL23 SYBR-Green primers (Rat)$25.00 USD$60.00 USD
RPL23 SYBR-Green primers (Chinese hamster)$25.00 USD$60.00 USD
RPLP0 SYBR-Green primers (Cow)$25.00 USD$60.00 USD
RPLP0 SYBR-Green primers (Pig)$25.00 USD$60.00 USD


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 ug of purified, linearized plasmid DNA that is directly ready for use in PCR.

Cloned Purified PlasmidsAccession #0.5 μg
0.5 µg Human RPLP0 Cloned qControlNM_001002$195.00 USD
0.5 µg Mouse RPL23 Cloned qControlNM_022891$195.00 USD
0.5 µg Rat RPL23 Cloned qControlNM_001007599$195.00 USD
0.5 µg Ch. Hamster RPL23 Cloned qControlNot Assigned$195.00 USD
0.5 µg Cow RPLP0 Cloned qControlNM_001012682$195.00 USD


A map of the plasmid clone is shipped with the control.

Exogenous Reporter Gene Positive Controls

Reporter 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 Rinella) 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.

Product1nm5nm
EGFP-S1 DS Positive Control$60.00 USD$150.00 USD
FLuc-S1 DS Positive Control$60.00 USD$150.00 USD
RLuc-S1 DS Positive Control$60.00 USD$150.00 USD


Universal Negative Control

Negative 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. The EGFP and FLuc DsiRNAs may also be used as negative controls if functional, targeting duplexes are desired (see above).

Product1nm5nm
DS Scrambled Neg$60.00 USD$150.00 USD
DS NC1$60.00 USD$150.00 USD
 
 
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