| First Author | Gordeeva O | Year | 2016 |
| Journal | MGI Direct Data Submission | Mgi Jnum | J:230330 |
| Mgi Id | MGI:5758772 | Citation | Gordeeva O (2016) Expression of the genes of the melanoma antigen (Mage) families in E7.5 mouse embryo. 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 clarify a consistency between the Mage expression patterns of the early embryo at mid-gastrula stage (E7.5) and the mouse embryonic stem cells differentiating in vitro, a comparative analysis of the Mage gene expression patterns was performed using quantitative real-time polymerase chain reaction (qRT-PCR) and RT-PCR. The qRT-PCR analysis revealed that the Mage expression pattern of the early mouse embryo at the mid-gastrula stage has considerable similarity to that of heterogeneous differentiated mouse embryonic stem cell populations containing the undifferentiated cells and progenitors of three germ layers. Embryonic and extraembryonic tissues of mouse embryos at E7.5 stage were obtained and cDNA prepared from total RNA. PCR assays were developed to reference sequences from the GenBank. Methods RNA extraction and reverse transcription Tissues from the embryos of C57BL/6 mice at E7.5 stage were recovered from uterine and placed on ice. Total RNAs from 2 embryos 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 TURBO DNase (Ambion/Invitrogen) according to the manufacturer's recommendations. cDNAs were synthesized using 1 microM oligo-dT18 primer and 0.1 mM dNTP mix (Fermentas), 2 micrograms of total RNAs and 200U of reverse transcriptase SuperScript III (Invitrogen) according to the manufacturer's 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 50-100 ng of total RNA was carried out in PCR mix containing 70 mM Tris-HCl buffer, pH 8.6 / 25°C with 16.6 mM (NH4)2SO4, 2.5 mM MgCl2, 1.25 U Colored Taq DNA polymerase, 0.1 mM cresol red, (E0211, Silex, Russia), 0.1 mM dNTPs (Fermentas), 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 94 degrees C for 5 min, each PCR cycle (35 cycles) consisted of 20 sec denaturing (94 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 and tris-borate buffer (x0.5, pH 8.3), stained with ethidium bromide and registered using transilluminator (BioRad, USA). |
