A technique of mRNA extraction and labeling from circulating lymphocytes of children treated with growth hormone replacement therapy for microarray analysis.

ABSTRACT

GH replacement therapy exhibits a wide spectrum of response in terms of growth. Nevertheless, standardized doses are still given in clinical practice. In order to optimize the therapy, it is necessary to identify its markers of responsiveness. Given the presence of GH receptors in the circulating lymphocytes, accessible by means of a simple blood withdrawal, blood becomes the tissue of choice as a source of RNA for in vivo gene expression analysis. Hence, the purpose of the present paper is to develop a method of preparation of RNA from lymphocytes suitable for microarray analysis, focusing on the reduction of the blood volume withdrawal in order to perform the analysis on pediatric subjects. After lymphocyte isolation and total RNA extraction from 6 ml of blood, we carried out an amplification procedure preserving the relative abundance of each transcript. Thereafter, we hybridized the labeled amplified RNA on an oligo chip (Human 30K A, MWGBiotech), but the unsuccessful detection of a good signal to noise ratio indicates that labeled RNA is still insufficient. Therefore, we suggest performing pools of total RNA from different subjects with similar responsiveness to the therapy. It can be speculated that, upon comparison of the obtained data with those derived from pools of controls properly responding to the therapy, specific hallmarks of the condition of low responsiveness, devoid of inter-individual variability, will be evidenced.