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Selection of housekeeping genes for gene expression studies in the adult rat submandibular gland under normal, inflamed, atrophic and regenerative statesAbstract: The program NormFinder identified the suitability of HPRT to use as a single gene for normalisation within the normal, inflamed and regenerative states, and GAPDH in the atrophic state. For normalisation to multiple housekeeping genes, for each individual state, the optimal number of housekeeping genes as given by geNorm was: ACTB/UBC in the normal, ACTB/YWHAZ in the inflamed, ACTB/HPRT in the atrophic and ACTB/GAPDH in the regenerative state. The most stable housekeeping gene identified between states (compared to normal) was UBC. However, ACTB, identified as one of the most stably expressed genes within states, was found to be one of the most variable between states. Furthermore we demonstrated that normalising between states to ACTB, rather than UBC, introduced an approximately 3 fold magnitude of error.Using NormFinder, our studies demonstrated the suitability of HPRT to use as a single gene for normalisation within the normal, inflamed and regenerative groups and GAPDH in the atrophic group. However, if normalising to multiple housekeeping genes, we recommend normalising to those identified by geNorm. For normalisation across the physiological states, we recommend the use of UBC.Saliva is secreted by three pairs of major salivary glands – parotid, submandibular and sublingual – as well as numerous other minor salivary glands located around the mouth. Understanding atrophy and regeneration of salivary glands is clinically important, and studies have shown that acinar cells, which are lost from atrophic salivary glands, re-differentiate from remaining duct cells [1-3]. Studies have also shown that mitotic proliferation of remaining acinar cells participates in the increase of acini in the regenerative process of parotid glands where acinar cells remain [4,5], and newly differentiated acinar cells proliferate actively in the regeneration of atrophic parotid glands in the absence of residual acinar cells [6]. In the submandibular gland, which differs histologically
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