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Rapid Cycle Real-Time (kinetic) PCR using LightCycler instumentation (Roche)

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Quantitative RT-PCR

In quantitative real-time Reverse Transcriptase-PCR (RT-PCR) normalization is performed using housekeeping genes as references against the expression level of a gene under investigation. The housekeeping genes are a large group of genes that code for proteins whose activities are essential for the maintenance of cell function. Roche offers 5 different LightCycler Housekeeping Gene sets to provide suitable reference genes for an individual relative quantification approach based on the estimated relative abundance of the target gene in the sample.

1) β2-Microglobulin (β2M) is a housekeeping gene which is detectable in a broad range of tissues, is relatively stably expressed and without pseudogenes known so far (9,10,11). It was shown, however, that in breast cancer cells its mRNA is rapidly degraded (12). β2-Microglobulin housekeeping gene belongs to the medium abundance class of mRNAs (13).

2) Glucose-6-phosphate dehydrogenase (G6PDH) is ubiquitously expressed and belongs to the class of “housekeeping” genes. Although ubiquitous, one issue that has to be taken into consideration upon choosing G6PDH RNA as a standard for relative quantitation is that the expression of G6PDH is not uniform. Basal activity varies from one tissue to another. In individual cell types (liver, adipose, lung, and proliferating cells), the cellular level of G6PDH is regulated by a number of stimuli (14,15,16,17). G6PDH housekeeping gene belongs to the medium abundance class of mRNAs (13).

3) 5-aminolevulinate synthase (ALAS) catalyzes the condensation of glycine with succinyl-CoA to form aminolevulinic acid. This nuclear-encoded mitochondrial enzyme is the first and rate-limiting enzyme in the mammalian heme biosynthetic pathway. The ubiquitous isoform (ALAS1) controls the rate of heme synthesis in non-erythroid mammalian tissues. The ALAS1 gene is reported as a constitutively expressed housekeeping gene (18). Alterations in steady state levels of ALAS mRNA have been reported. Stimulating effects have been described for cAMP, inhibitory effects for phorbol esters and insulin(19, 20).

4) Hypoxanthinephophoribosyltransferase (HPRT) plays an important role in the purine salvage pathways, where it mediates the recycling of hypoxanthine and guanine into the usable nucleotide pools. The HPRT gene is reported as a constitutively expressed housekeeping gene (21). HPRT RNA levels are very low, 1 to 10 molecules per cell (22) which makes it suitable as an endogenous mRNA control in RT-PCR for highly sensitive quantification of low copy or rare mRNAs (23-25). Due to the existence of pseudogenes (24), the assay requires that the primer and probes are chosen carefully to avoid amplification or detection of the pseudogenes from contaminating DNA which might be present in the RT-PCR assay. HPRT mRNA levels are higher in growing cells (22). Reduced levels of HPRT mRNA are observed in cells from indivduals carrying inherited mutations in the HPRT locus (26,27). Mutations in the HPRT gene are responsible for a wide spectrum of diseases like HPRT hyperuricemia, HPRT-related neurologic dysfunction and Lesch-Nyhan disease. Mutations in the HPRT gene can be induced in response to drug treatment (28-30) or by X-irradiation (31). These influences may cause deletions or alterations leading to splice variants.

5) Porphobilinogen deaminase (PBGD) is encoded by two distinct mRNA species expressed in a tissue-specific manner from a single gene. One transcript is expressed only in erythroid tissues, while the housekeeping transcript that is used in this set is expressed in all tissues (32-34). The PBGD housekeeping gene is expressed in a broad range of tissues at a relatively stable expression level and without known pseudogenes thus far. For more informations please refer to (5, 6, 35-37). Although PBGD expression levels are comparable in various tissues, they may vary in clinical samples (37). PBGD housekeeping gene belongs to the low abundance class of mRNAs (37, 13).

For more detailed information and specific references: Housekeeping Gene Selection

To obtain the value of the target under investigation and the value of the housekeeping gene in the same sample, a standard curve is used. Then the absolute concentration of the target is divided by the absolute concentration of the housekeeping gene. The resulting target/reference ratio expresses the amount of target now normalized to the level of the reference gene within each unknown sample. Fold changes of “treated” vs “untreated” samples are determined by dividing the above ratios for the target pair.