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Validating internal controls for quantitative plant gene expression studies

Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression.

We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied.

Studies in mammalian and microbial systems, where real-time q RT-PCR has been most extensively applied to date, have begun to include evaluations of various housekeeping genes for normalization [7-11]. [7] recognized the importance of using statistical approaches to selecting the best internal controls for a given set of samples, and developed a procedure to select internal controls based on the mean pairwise variation of a gene from all other tested control genes.

Until recently, internal controls (often referred to as housekeeping or maintenance genes), were selected based on stability of expression in qualitative studies (e.g., visual examination of RNA gel-blots), via low-sensitivity assays such as densitometry of hybridized blots, or via semi-quantitative RT-PCR.

None of these will be adequate for identifying reliable internal controls for real-time q RT-PCR.

CONCLUSION: Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression.

The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability.

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