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INTEGRATEDDNATECHNOLOGIESPRODUCTSFUNCTIONAL GENOMICSInternal 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 differenthousekeeping genes have been used for this purpose (such as β-Actin, GAPDH, or Cyclophilin), most of these genes showfluctuation in expression levels with different treatments and are not as invariant as is needed for a true internal qRT-PCRcontrol. IDT has developed the following SYBR® Green assays suitable for use as an internal normalization standard in qRT-PCRanalysis (ΔΔCt method). When combined with use of the cloned copy number control plasmids, these reagents permit relativeRNA mass-loading normalization plus absolute quantitative PCR to be performed.ProductRPLP0 SYBR®-Green Primers (Human) - 1 nmole or 5 nmolesRPL23 SYBR®-Green Primers (Mouse) - 1 nmole or 5 nmolesRPL23 SYBR®-Green Primers (Rat) - 1 nmole or 5 nmolesRPL23 SYBR®-Green Primers (Chinese hamster) - 1 nmole or 5 nmolesRPLP0 SYBR®-Green Primers (Cow) - 1 nmole or 5 nmolesRPL4 SYBR®-Green Primers (Pig) - 1 nmole or 5 nmolesIt 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 numberstandards permit true quantitative measurements to be made. The assay amplicon for each of the above positive control qRT-PCR reactions has been cloned into pBlueScript TM -II and sequence verified. Clones are provided as 0.5 µg of purified, linearizedplasmid DNA that is directly ready for use in PCR.Cloned Purified PlasmidsRPLP0 Cloned qControl (Human) - 0.5 µgRPL23 Cloned qControl (Mouse) - 0.5 µgRPL23 Cloned qControl (Rat) - 0.5 µgRPL23 Cloned qControl (Chinese hamster) - 0.5 µgRPLP0 Cloned qControl (Cow) - 0.5 µgRPL4 Cloned qControl (Pig) - 0.5 µgAccession#NM_001002NM_022891NM_001007599Not assignedNM_001012682DQ845176Visit www.idtdna.com for a map of the plasmid clones in PDF format.©2008 Integrated DNA Technologies www.idtdna.com21