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PAGE  PAGE 8 Electronic Supplementary Material ESM-Methods Real-time quantitative PCR and western blot analysis We used the TrizolTM reagent (Invitrogen, USA) and followed the manufacturer’s instructions to isolate total RNA  ADDIN EN.CITE EndNoteCiteAuthorKrivoruchko/AuthorYear2010/YearRecNum290/RecNumDisplayText[1]/DisplayTextrecordrec-number290/rec-numberforeign-keyskey app=EN db-id=pz2zzt52otvz0xedzs7pe9xsxr0f5zasea2w290/key/foreign-keysref-type name=Journal Article17/ref-typecontributorsauthorsauthorKrivoruchko, Anastasia/authorauthorStorey, Kenneth B/author/authors/contributorstitlestitleRegulation of the heat shock response under anoxia in the turtle, Trachemys scripta elegans/titlesecondary-titleJournal of Comparative Physiology B/secondary-title/titlesperiodicalfull-titleJournal of Comparative Physiology B/full-title/periodicalpages403-414/pagesvolume180/volumenumber3/numberdatesyear2010/year/datesisbn0174-1578/isbnurls/urls/record/Cite/EndNote[ HYPERLINK \l _ENREF_1 \o Krivoruchko, 2010 #290 1]. The purity and concentration of each RNA sample was estimated using the Nanodrop 2000/2000C (Thermo Scientific). Approximately 1 μg of total RNA from each sample was used to generate first-stand cDNA using an oligo-dT (15) primer (Promega) and M-MLV reverse transcriptase (Promega) according to the manufacturers protocol. Fluorescent real-time quantitative PCR was performed in a 10-μL total reaction volume comprising 5 μL of a premix (BIO-RAD, SsoFast TMEvaGreen?Supermix), 0.5 μL of each primer (2 μM) for quail or 1 μL of each primer (2 μM) for other species, 1 μL of cDNA template, and water. PCR was conducted using a Mx3000P detection system (Stratagene) and the parameters were as follows: 30 s at 95°C, followed by 35 cycles of 10 s at 95 °C, 10 s at 60 °C, and 25 s at 72 °C (for P. sinensis); or 32 cycles of 10 s at 95 °C, 10 s at 59 °C, and 25 s at 72 °C (for T. septentrionalis); or 35 cycles of 10 s at 95 °C, 10 s at 60 °oC, and 5 s at 72 °C (for C