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Application of MIQE Guidelkines to Measuring Effects of Maternal Smoking on nAChR mRNA Expresiion in the Striatum
Author(s) -
Williamson Paula Denise,
Weiss Emily Sarah,
Middleton Courtney,
Adams Jamie,
Dolan Maureen C,
Buchanan Roger A
Publication year - 2011
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.25.1_supplement.lb54
Subject(s) - striatum , complementary dna , messenger rna , primer (cosmetics) , nicotinic acetylcholine receptor , gene expression , medicine , endocrinology , biology , microbiology and biotechnology , andrology , nicotinic agonist , chemistry , receptor , gene , biochemistry , organic chemistry , dopamine
Prenatal exposure to the constituents of cigarette smoke (maternal smoking) is known to have deleterious effects on health. We report effects of maternal smoking on expression of nicotinic acetylcholine receptor (nAChR) subunits in the striatum. Pregnant dams were exposed to cigarette smoke (350 ml/exp, 3 exp/day) from gestational day 4 through parturition. Brain regions were harvested from male and female prepubertal (PND 20), pubertal (PND 35) and post‐pubertal (PND 60) pups of unexposed and exposed dams. RNA was extracted from tissue samples using a Promega Maxwell 16.. cDNA was reverse‐transcribed using iScript Select cDNA synthesis and amplified and detected with SSO Fast EvaGreen Supermix Green by real time RTqPCR. cDNA was pooled and used to evaluate primer efficiencies, and reference gene stability. Primer efficiencies ranged from 72 to 117% and reference gene stabilities (M values) ranged from 0.2 to 0.8. mRNA expression of nAChR subunits α 2 , α 3 , α 4 , α 5 a 7 , β 2 and β 4 was measured and compared at each PND, exposure and gender. Differences in expression of several subunits were observed. MIQE standards for reporting results of RT‐qPCR experiments were followed. We also report results of mRNA extraction experiments performed on very small amounts of CNS tissue. These results show that sufficient amounts of mRNA can be obtained from samples as small as 1 mm 3 and from identified brain regions from newborn (PND 1) pups.