PMC:3475480 / 6302-7742 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3475480","sourcedb":"PMC","sourceid":"3475480","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3475480","text":"RNA extraction and real-time PCR\nRNA was extracted from the tissues of siRNA-treated mice with TRIzol (Invitrogen). We synthesized cDNA from 500 ng of total RNA using the PrimeScript™ RT reagent kit (TaKaRa, Otsu, Japan) per the manufacturer's protocol. Casz1 mRNA was analyzed by real-time PCR. One-tenth of the cDNA reaction was added to a final volume of 20 µL for each reaction containing SYBR Green I (TaKaRa). The primers for Casz1 and glyceraldehye-3-phosphate dehydrogenase (GAPDH) were 5'-GTCTCTTCGGGAACTGCAAG-3'/5'-TGGGACACAGGCACTGTAGA-3' and 5'-GCTCTCTGCTCCTCCTGTTC-3'/5'-CAATACGACCAAATCCGTTG-3', respectively (forward/reverse sequence).\nQuantitative real-time PCR was performed on an ABI Step One Real-time PCR system (Applied Biosystems, Foster, CA, USA) using the following program: 45 cycles of 95℃ for 10 s, 60℃ for 15 s, and 72℃ for 20 s. To normalize the amount of sample cDNA in each reaction, the Ct value of each target gene was subtracted by that of GAPDH to obtain delta Ct [14]. To calculate the fold-change in expression, the delta Ct value of the case sample was subtracted by that of the control (delta delta Ct = delta Ct case - delta Ct control).\nReverse-transcription PCR was performed similarly to quantitative real-time PCR, except that it used 35 cycles for amplification. The PCR products were separated by electrophoresis in a 2.0% agarose gel and visualized under UV lamp after ethidium bromide staining.","divisions":[{"label":"Title","span":{"begin":0,"end":32}}],"tracks":[{"project":"2_test","denotations":[{"id":"23105927-11846609-44845687","span":{"begin":998,"end":1000},"obj":"11846609"}],"attributes":[{"subj":"23105927-11846609-44845687","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93b1","default":true}]}]}}