Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

doi:10.1186/1743-7075-4-26

Nutrition & Metabolism

Volume 4

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Romero MM
Grasa MM
Esteve M
Fernández-López JA
Alemany M

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Grasa MM
Esteve M
Fernández-López JA
Alemany M
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Methodology

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Maria del Mar Romero¹^,2 , Maria del Mar Grasa¹^,2 , Montserrat Esteve¹^,2 , José Antonio Fernández-López¹^,2 and Marià Alemany¹^,2

¹Department of Nutrition and Food Science, Faculty of Biology, University of Barcelona, Barcelona, Spain

²Ciber Fisiopatologia Obesidad y Nutrición (CB06/03) Instituto de Salud Carlos III, Madrid, Spain

author email corresponding author email

Nutrition & Metabolism 2007, 4:26doi:10.1186/1743-7075-4-26


Published:	26 November 2007

Abstract

Background

Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions.

Methods

With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell.

Results

The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation.

Conclusion

The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.