Despite the popularity of quantitative PCR, there are many factors that the researcher must consider when planning experiments and one of these is to normalise for variations in the amount of template material. An ideal control is one that is unaffected by all conditions or treatments. Several strategies for normalisation have been proposed including the use of total input RNA, cell number and rRNA. The most popular method is normalisation to an appropriate internal reference gene.
Quantace has developed a Mouse Normalisation Gene Panel using SYBR® Green I chemistry. The panel comprises primer sets for 12 candidate reference genes from functionally diverse families (see Table below).
Primer
pair | Cat.no. | Gene | Ensemble id / genbank | Product
spans
exons | | SSDHA | QT12X
MS-01 | Succinate
dehydrogenase
complex,
subunit A | ENSMUSG00000021577 | 14 and 15 | | GACTIN | QT12X
MS-02 | Gamma actin,
cytoplasmic | ENSMUSG00000062825 | 5 and 6 | | YWHAZ | QT12X
MS-03 | tyrosine 3-monooxygenase, zeta polypeptide | ENSMUSG00000022285 | Not intron
spanning | | RPS18 | QT12X
MS-04 | Ribosomal
Protein S18 | ENSMUSG00000008668 | 1 and 2 | | GAPDH | QT12X
MS-05 | Glyceraldehyde-
3-phosphate | ENSMUSG00000057666 | 4 and 5 | | B2MG | QT12X
MS-06 | Beta-2
microglobulin | ENSMUSG00000060802 | 2 and 3 | | RPL13A | QT12X
MS-07 | Ribosomal
protein L13a | ENST00000270634 | 10 and 11 | | PGK | QT12X
MS-08 | Phosphoglycerate
kinase 1 | ENSMUSG00000062070 | 9 and 10 | | CALNX | QT12X
MS-09 | Calnexin | ENSMUSG00000020368 | 1 and 2 | | BACTIN | QT12X
MS-10 | Beta actin
cytoplasmic | ENSMUSG00000029580 | 3 and 4 | | RN18S | QT12X
MS-11 | 18S rRNA | Genbank (X56974) | intronless
gene | | UBQC | QT12X
MS-12 | Ubiquitin C | ENSMUSG00000008348 | 1 and 2 |
Identification of the most appropriate reference gene is achieved by comparing the gene of interest against the Mouse Normalisation Panel, using standard analysis packages such as GeNorm (Vandesompele et al. 2002), BestKeeper (Pfaffl et al. 2004) or Normfinder (Andersen et al. 2004). These primer sets have been optimised for use with the SensiMix range of reagents. The use of SensiMix with this gene panel results in amplification efficiencies of between 95 and 102% along with excellent R, R2 and M values and an absence of primer dimers.
These primers can be ordered as a set of 12 or as individual primer sets. An application note containing ideal results is supplied with the full panel but can be downloaded as a pdf file here. | House-Keeping Genes | Pack Size | Ref.: | Mouse Normalisation
Gene Panel | 12 x 100 rxns | QT12X-MS | | Mouse Gene, SSDHA | 100 rxns | QT12X-MS-01 | | Mouse Gene, GACTIN | 100 rxns | QT12X-MS-02 | | Mouse Gene, YWHAZ | 100 rxns | QT12X-MS-03 | | Mouse Gene, RPS18 | 100 rxns | QT12X-MS-04 | | Mouse Gene, GAPDH | 100 rxns | QT12X-MS-05 | | Mouse Gene, B2MG | 100 rxns | QT12X-MS-06 | | Mouse Gene, RPL13A | 100 rxns | QT12X-MS-07 | | Mouse Gene, PGK | 100 rxns | QT12X-MS-08 | | Mouse Gene, CALNX | 100 rxns | QT12X-MS-09 | | Mouse Gene, BACTIN | 100 rxns | QT12X-MS-10 | | Mouse Gene, RN18S | 100 rxns | QT12X-MS-11 | | Mouse Gene, UBQC | 100 rxns | QT12X-MS-12 |
* 1 reaction = 1µl in 50 µl total volume |
