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These novel ICG could be used for normalization in similar studies as alternatives to the less-reliable ACTB, GAPDH, or B2M.
However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization.
Thus, the present study was aimed to assess the appropriateness of the commonly used RGs and to develop a set of genes that can be used for normalization of qPCR gene expression data in the profiling of gene expression in C. batrachus under control and experimental (hypoxic) conditions.
In the study of Meuleman, Engwegen et al. [14], they compared various different algorithms that can be used for normalization.
Thus, reference genes that present with ΔCT values ≥ 1.0 should not be used for normalization, if indeed the observed changes in gene expression are due to biological versus technical variance, which may be difficult to discern in some circumstances.
For example, in a study by Peltier et al[18] that carefully evaluated several candidate miRNAs for stability of expression across five types of cancer and normal tissues by RT-PCR, miR-191 was so stably expressed that it was proposed as a gene to be used for normalization.
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GAPDH was used for normalization.
GAPDH was used for normalization of expression.
A pool of all the samples was used for normalization.
GAPDH was used for normalization of the genes of interest.
GAPDH was used for normalization of RT-qPCR data.
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