PMC:4502369 / 13610-15778
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4502369","sourcedb":"PMC","sourceid":"4502369","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502369","text":"RT-PCR and sequencing\nFor analysis of gene expression with RT-PCR, RNA of the testis and oviduct samples from two controls and ISTS homozygous animals were extracted (RNeasy Midi kit; Qiagen). Total RNA was reverse-transcribed with random primers and an RT-PCR kit (ImProm-II Reverse Transcription System; Promega) according to the manufacturer’s instructions. Synthesized cDNA was amplified using gene-specific primers (Supporting Information, Table S1). The housekeeping gene ribosomal S18 (RIBS18) was used as a reference gene to calculate the relative expression. cDNA samples were diluted to 20 ng/μl prior to use. The qPCR was performed with a ViiA 7 Real-Time PCR System in 96-well microtiter plates using Absolute qPCR SYBR Green ROX Mix (VWR). Amplification by qPCR contained 12.5 μl of Absolute qPCR SYBR Green Mix, 100 ng of cDNA, and 70 nM of each primer in a final volume of 25 μl. Amplifications were initiated with 15 min of enzyme activation at 95°, followed by 40 cycles of denaturation at 95° for 15 sec, primer annealing at 60° for 30 sec, and extension at 72° for 30 sec. All samples were amplified in triplicate, and the mean value was used in further calculations. Raw data were analyzed with the sequence detection software (Applied Biosystems) and relative quantitation was performed with GenEx software (MultiD). Ratios between the target and reference gene were calculated using the mean of these measurements. A standard curve for each primer pair was produced by serially diluting a control cDNA and used to correct for differences in amplification. A melting curve analysis was performed allowing single product-specific melting temperatures to be determined. No primer–dimer formations were generated during the application of 40 real-time PCR amplification cycles.\nFor sequencing, the RT-PCR amplicons were purified using ExoSAP-IT (Amersham Biosciences). PCR fragments were sequenced in both directions with the same primers used for amplification. Sequencing was performed on a MegaBace 500 capillary DNA sequencer (Amersham Biosciences) using DYEnamic ET Terminator Kits with Thermo Sequenase II DNA Polymerase (Amersham Biosciences).","divisions":[{"label":"title","span":{"begin":0,"end":21}},{"label":"p","span":{"begin":22,"end":1795}}],"tracks":[]}