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Expression of one‐carbon metabolism genes in cultured primary lymphocytes
Author(s) -
Zhu Jie,
Kohlmeier Martin
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.647.2
Subject(s) - housekeeping gene , gene expression , gene , microbiology and biotechnology , vacutainer , biology , peripheral blood , complementary dna , chemistry , immunology , biochemistry , chromatography
The aim of the experiments was to measure gene expression in lymphocytes from peripheral blood. This procedure is to be used for assessing the impact of genomic variants on gene function. Lymphocytes were isolated from venous human blood and then cultured with phytohemagglutinin (PHA). Optimal growth was achieved with 0.8 μg/ml of purified PHA (Remel Europe, UK) resulting in about six fold expansion after 9 days. Both lower and higher PHA concentration induced proliferation less effectively. Expression of MTHFR, MTHFD1 and DHFR was measured in transcribed cDNA with validated TaqMan assays (Applied Biosystems, CA). The RNA obtained from less than one million cultured cells was sufficient for multiple expression experiments. Expression of all three targeted genes, normalized to a housekeeping gene, was approximately constant between days 6 and 10. These experiments demonstrate that gene expression analysis of lymphocytes from modest amounts of peripheral blood (a single vacutainer CPT tube with 5 ml blood) is feasible and practical.