| First Author | Gordeeva O | Year | 2018 |
| Journal | MGI Direct Data Submission | Mgi Jnum | J:255000 |
| Mgi Id | MGI:6113847 | Citation | Gordeeva O (2018) Expression of the genes of the melanoma antigen (Mage) families in brain. MGI Direct Data Submission |
| abstractText | Olga Gordeeva Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, Moscow, 119334, Russia Summary To understand an involvement of Mage (Melanoma antigen) genes in somatic organ development the expression profiles of Mage gene families was studied in the midbrains and hindbrains of C57BL/6 mouse embryos at the E10.5, E14.5 and E19.5 developmental stages using the RT-PCR analysis. High expression of Mage-d1, -d2, -e1, -e2, -h1, -l2 genes (II class of Mages) was detected in the midbrains and hindbrains at all studied stages while the expression of Mage-a and Mage-b family genes (I class of Mages) varied considerably. Mage-a family genes, Mage-a2, -a4, -a6, -a8 and Mage-a10, were found to be expressed in the midbrains and hindbrains at different levels. The expression of 5 of 6 genes of Mage-b family (Mage-b1, -b3, -b4, -b16, -b18) was detected in the midbrains and hindbrains while no expression of Mage-b5 was detected in studied brain structures at the E10.5, E14.5 and E19.5 developmental stages. Thus, the expression of most genes of the Mage families (16 of 17) was detected in the brain anlages of mouse embryos at the E10.5, E14.5 and E19.5 developmental stages. Moreover, the expression patterns of I class of Mage genes varied between different stages while no differences were found in the expression patterns of II class of Mage genes. Methods RNA extraction and reverse transcription Brain tissues from the C57BL/6 mouse embryos at E10.5, E 14.5 and E19.5 stages were isolated and placed on ice. Total RNAs from embryonic brain anlages (1/2 - 2 per sample, n=3 samples) were extracted using the TRIzol Reagent (Invitrogen).The RNA yield and quality were analyzed by NanoDrop 2000 (Thermo Scientific). Each sample was treated with dsDNase (Thermo Scientific/Fermenras) according to the manufacturer's recommendations. cDNAs were synthesized using 1microM oligo-dT18 primer and 0.1 mM dNTP mix (Thermo Scientific/Fermenras), 2 micrograms of total RNAs and 200U of Maxima Reverse Transcriptase (Thermo Scientific/Fermenras) according to the manufactures protocols. The expression of reference gene hypoxanthine guanine phosphoribosile transferase Hprt was used for normalization of cDNAs. Primer design and PCR PCR primers of 18-26 bp in length with a 40-60% GC content and Tm of 60 degrees C plus/minus 2 degrees C were selected using the Beacon Designer software (PREMIER Biosoft, USA) and PRIMER3 (Whitehead Institute/MIT Center for Genome Research, Cambridge, MA) to amplify products of 75-200 bp in length. Primers were pre-screened to determine the optimal conditions for specific cDNA amplification using adult mouse testes cDNAs as a positive control. They were tested using 30 PCR cycles at 60 degrees C annealing temperature under standard assay conditions (see below). PCR amplification Hot-lid PCR amplification of cDNA equivalent to 100 ng of total RNA was carried out in PCR mix containing 20 mM ris-HCl buffer (pH 8.6 / 25°C) with 5 mM (NH4)2SO4, 20 mM KCl and 2.5 mM MgCl2, 0.25 U Maxima Hot Start Taq DNA polymerase (Thermo Scientific/Fermenras), 0.1 mM dNTPs (Thermo Scientific/Fermenras), and 0.2 microM of each primer were used at each reaction. PCR analysis of gene expression was carried out on an Eppendorf master cycler (Eppendorf, Germany) according to following program: preliminary denaturation at 95 degrees C for 5 min, each PCR cycle (37 cycles) consisted of 20 sec denaturing (95 degrees C), 20 sec annealing of primers (60 degrees C), and 30 sec elongation (72 degrees C) and final extension at 72°C for 10 min. All assays were conducted under identical conditions. The PCR products were then separated on a 1.5% agarose gel (TopVision Agarose, Fermentas) and Tris-borate buffer (x0.5, pH 8.3), stained with ethidium bromide and registered using transilluminator (BioRad). |
