Validating internal controls for quantitative plant gene expression

") Dissociation curve analysis of primer specific products was performed by constantly monitoring the fluorescence with increasing temperatures from 60 to 95 °C.

Melt curves were generated by plotting the negative first derivative of the fluorescence (−d/d Figure 3 Sequence identity of the PCR products for the five reference genes evaluated in this study.

Expression analysis of two lung specific markers, surfactant protein C (Sftp C) and Clara cell-specific 10 k DA protein (Scgb1a1), normalized to each of the five reference genes tested here, confirmed our results and showed that incorrect reference gene choice can lead to artefacts.

Moreover, a combination of two internal controls for normalization of expression analysis during lung development will increase the accuracy and reliability of results.

Microarray expression profiling in rice has not been widely reported with relatively few publicly available data.

Rice ( L.) is an important crop and the most advanced model for monocotyledonous species; its nuclear genome has been sequenced and molecular tools are being developed for functional analyses.

However, high-throughput methods for rice research are still limited and a large-scale q RT-PCR platform for gene expression analyses has not been reported.

Studies using q RT-PCR have also not been widely reported, have focussed on small groups of genes, and in many cases were only used to confirm expression changes from microarray experiments.

Therefore, the utility of q RT-PCR as a high-throughput method in rice has not been investigated.

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The stability of expression of five different reference genes (Tuba1a, Actb Gapdh, Rn18S and Hist4h4) was calculated within five experimental groups using the statistical algorithm of ge Norm software.

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