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Summer Research Fellowship Programme of India's Science Academies

Expression of AHR Gene in Human Microglial Cells

Gade Kalyani

Department of Pharmacy, Koneru Lakshmaiah Education Foundation (K L deemed to be university), Vaddeswaram, Guntur, Andhra Pradesh, India 522502

Sunit K. Singh

Laboratory of Human Molecular Virology & Immunology, Molecular Biology Unit, Faculty of Medicine, Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi 221005, India.

Abstract

The objective of this study was to carry out the expression of AHR gene using CHME3 human microglial cells. Methods include DMEM media preparation, thawing, passaging, and trypsinization. Cell counting was done at 20 %, 60 % confluency at passage P8 level and cryopreservation followed by aliquoting the cells. The labelled cryovials were later transferred into liquid nitrogen (-196 º C) container until further use. The RNA isolation was carried out using Trizol method which includes cell lysis, homogenisation, Phase separation, RNA precipitation, and quantification. The isolated total RNA obtained using 2 samples was found to be of good quality with concentrations 319 ng/μl and 269 ng/μl, A260/280 ratio was 2.07 along with A260/230 ratios were 1.66 and 1.67 respectively . This isolated RNA was used for the synthesis of cDNA using the thermal cycler and RT enzymes. The cDNA sample obtained was estimated by End point PCR using primer pair and buffer mixture which was further analysed for the expression of AHR gene. Gel electrophoresis was further obtained at band scale 100bp to find out the desired gene expression. Since, AHR is involved in inducing both pro-inflammatory and anti-inflammatory responses in activated microglial cells, these were further expressed in CHME3 human microglial cells in the present study.

Keywords: Cryopreservation, DMEM, cDNA, AHR, Polymerase chain reaction.

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