The aim of the study is to evaluate oxygen regulated gene expression in human peripheral blood lymphocytes using microarray analysis. The results of this study are expected to provide useful marker transcripts whose expression is highly affected by hypoxia for further hypoxic gene expression studies using peripheral blood lymphocytes. Keywords: peripheral blood lymphocytes, hypoxia-regulated transcripts
Overall design
The lymphocyte isolation and incubation at 1% or 20% Oxygen tensions are explained in detail in the methods section of Smith TG, Brooks JT et al., (2006) Mutation of von Hippel-Lindau Tumour Suppressor and Human Cardiopulmonary Physiology PLoS Medicine 3, e290. Briefly, following venesection, 40 ml of blood was drawn from a human volunteer. Peripheral blood lymphocytes were isolated from the blood sample by centrifugation over density gradient medium (Ficoll-Paque plus, Amersham Biosciences UK) followed by incubation at 37 degrees Celsius to allow for monocyte adhesion. The isolated lymphocytes were then divided into two aliquots which incubated at either 20% or 1% ambient oxygen tensions at 37 degrees Celsius for 20 hours. Lymphocytes from each aliquot were then harvested by centrifugation and RNA was extracted from the lymphocytes using a column based extraction method (RNAqueous-4PCR, Ambion US). The entire procedure was repeated three times on the same volunteer to give 6 RNA samples from the three paired sets of hypoxic/normoxic incubations. Following in vitro transcription and incorporation of biotin-16 UTP into the amplified transcripts, the six samples were then analysed for transcript expression on a Sentrix Human 6-Expression BeadChip (Illumina California US) platform. This platform contains six individual microarrays allowing all six samples to be analysed simultaneously on a single platform. The results from the three paired sets of hypoxic/normoxic exposures were used to statistically determine any transcripts showing hypoxic regulation in human peripheral blood lymphocytes.
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