| First Author | Gordeeva O | Year | 2016 |
| Journal | MGI Direct Data Submission | Mgi Jnum | J:237181 |
| Mgi Id | MGI:5811254 | Citation | Gordeeva O (2016) Expression of the genes of the melanoma antigen (Mage) families in placenta. 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 dynamics of Mage gene expression during mouse development, the expression pattern of Mage gene families was studied in the placentas of mouse embryos of C57BL/6, CBA and BALB/c strains at the E10.5, E14.5 and E19.5 stages. Analysis of Mage gene expression patterns in mouse placentas was performed using RT-PCR. The RT-PCR analysis revealed that Mage expression patterns were significantly similar between C57BL/6, CBA and BALB/c mouse placentas at all studied stages. Thus, no differences were found in the Mage gene expression patterns in placentas of mice with different genetic background and at the E10.5, E14.5 and E19.5 stages of development. PCR assays were developed to reference sequences of Mage genes of Mus musculus (house mouse) from the GenBank. Methods RNA extraction and reverse transcription Placenta tissues from the embryos of C57BL/6, CBA and BALB/c mice at E10.5, E 14.5 and E19.5 stages were isolated from embryos and other extraembryonic structures and placed on ice. Total RNAs from 1/2 -1/4 of placentas of 1 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 1microM oligo-dT18 primer and 0.1 mM dNTP mix (Fermentas), 2 micrograms of total RNAs and 200U of reverse transcriptase Protoscript II (New England Biolabs) 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 80-200 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 70 mM ????-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 (TopVision Agarose, Fermentas) and tris-borate buffer (x0.5, pH 8.3), stained with ethidium bromide and registered using transilluminator (BioRad, USA). |
