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Gene Expression on Rat1 Fibroblast Cells After Transformation byEVI1

Zeloso Muenhowossimbu Tiago 1*

Subject : Life Sciences Master´s dissertation : Gene Expression on Rat1 Fibroblast Cells After Transformation by EVI1 (GlasgowCaledonian University, UK); 2017 Keywords : EVI1; Gene expression; CAIII; Rat1neo; Rat15.6

AIM This research aimed to demonstrate the gene expression of Rat1 Fibroblast cells overexpressing EVI1 aswell as the relation between the levels of expression of the EVI1 transcription factor and CAIII gene expressionin the transformed cells and looking at the molecular and cellular mechanisms of EVI1.

MATERIAL AND METHODS To assess CAIII gene expression in relation to cellular environment EVI1 level, real-time quantitativepolymerase chain reaction (qPCR) was performed followed by Western blot to confirm the gene and proteinexpression in Rat1neo and Rat15.6 cells, including the effect of CAIII gene in Rat1neo after hydrogen peroxide(H 2 O 2 ) treatment and silencing the expression of CAIII in these cells by siRNAs.To confluent monolayers of Rat1neo and Rat15.6 fibroblasts on 6 well tissue culture dishes was added RP1Buffer and 1M of dithiothreitol (DTT) and transferred to Nucleospin®Filter Nucleospin®RNA/Protein (Macherey-Nagel Products, Neumann-Neander Str. Germany) rDNase was added, and the buffers RA2, RA3 washed thefilters. RNAse-free Water extracted the total cellular RNA, and PP buffer, ethanol 50%, and PBS-TCEB bufferwere used to extract the protein. The RNA levels were measured and analysed in nanodrop spectrophotometerEpoch device. The RNA concentration was adjusted to 200ng/µl for analytical agarose gel electrophoresis. TheRNA samples were added sample buffer of 10XMOPS, formaldehyde and formamide and loading buffer in afume hood. The samples were pre-heated at 65°C followed by a brief centrifuge and were run at 100V on a gelfor 20 minutes then photographed with a Benchtop 3UV TM Transilluminator device.cDNA was obtained by mixing from InvitrogenTM kit composed of nuclease free water, RT enzyme mix,2XRT reaction mix and RNA in a 0.2ml dome capped PCR tube and incubated in DNA thermal cycler in adifferent time before and after mix with RNAseH.RNA from cultured Rat1neo and Rat15.6 cells was obtained and an analytical agarose gel electrophoresiswas performed followed by cDNA preparation for qPCR. 2µl of cDNA of Rat1neo and Rat15.6 diluted in aquantity of 1 in 5 was prepared and mixed with 5’ and 3’ CAIII and GAPDH primers and probes and Nucleasefree water added. 30µl of PerfeCta FastMix II Hot Start QPCR (Quanta BioScienceTM Manchester, UK), andthis was finally amplified in the MJ QPCR DNA thermal cycler. The resulting data was processed in MicrosoftExcel where mean and standard deviation of Ct values (threshold cycle) were calculated.All antibodies and blocking were diluted in blotto (0.5 marvel and 10ml of 1XTBST) to prevent non-specificbinding. The primer and second antibodies were diluted in 1/1000 and 1/15000 respectively. Finally, the images.were acquired in Li-Cor Odyssey image analyser using image studio software version 2.0 in a black and whitebackground

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1 - Faculdade de Medicina do Huambo – Universidade Josê Eduardo dos Santos. Orcid: 0000-0002-3086-8885 * - Autor correspondente. Email: mzmuenho@hotmail.com Doi: https://doi.org/10.54283/RACSaude.2789-2832.v2n1_2021.p21-23 Recebido : 19 de Março de 2021 / Aceite : 2 de Maio de 2020 / Publicado 30 de junho de 2021

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