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Kyunghwa Jeong edited Methods_qpcr.tex
almost 9 years ago
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\subsection*{Quantitative real-time PCR}
Male adult flies were maintained in 4 days of 12h:12h LD cycle and following continuous dark period (DD).
120-150 120--150 fly heads were collected each time point (CT0-CT20) in second day of DD.
Total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo scientific).
PCR was performed using TOPreal qPCR 2x premix (RT500M, Enzynomics, South Korea). Expression levels of \emph{period} gene was normalized to rp49 levels.
Primer sets used: period (5'-GACCGAATCCCTGCTCAATA-3', 5'-GTGTCATTGGCGGACTTCTT-3'), rp49 (5'-ATGACCATCCGCCCAGCATA-3', 5'-GAGAACGCAGGCGACCGTTG-3')
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