Abi prism 7700 sequence detection system user bulletin 2

Total RNAs were extracted with the TRIzol Reagent (Invitrogen) from the whole body of male adult flies and then ribosomal RNAs were depleted from the total RNAs using the RiboMinus Eukaryote Kit (Invitrogen). With these coding and noncoding RNAs, the Illumina compatible strand-specific RNA-Seq protocol was used to make the cDNA library, which was then sequenced by Illumina NextSeq500 sequencer in the Genomics facility (http://research.njms.rutgers.edu/genomics/). The significance of the expression changes was evaluated by the Fisher’s exact test (p-value). For the RT-PCR experiment to check transcriptional expression of the genes, 5 μg of the total RNA purified from adult flies were treated with DNase I (RNase-free, Roche) and used to produce oligo dT-primed cDNAs (SuperScript II RT, Invitrogen). Then, the cDNAs were used as templates for quantitative real-time PCR that was performed with power SYBR green PCR mix (Applied Biosystems) [51 (link)]. The rp49 gene was used as an internal reference for normalizing the quality of total RNA purified from each fly. Expressional fold of the various genes was determined by the comparative C_T method (ABI Prism 7700 Sequence Detection System User Bulletin #2, Applied Biosystems).

To construct rpd3WT and rpd3ADA transgenes (Fig. 6), the rpd3 wild-type cDNA (1,563 bp) was cloned into the pCRII-TOPO vector (Invitrogen) and, from the rpd3WT plasmid the rpd3ADA clone (S419A/S421A) was produced using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). After sequencing was completed, the plasmids were subcloned into a XhoI/XbaI digested pUASTattB vector.
To check transcriptional expression of the genes, 5 μg of the total RNA purified from adult flies (TRIzol, Invitrogen) were treated with DNase I (RNase-free, Roche) and used to produce oligo dT-primed cDNAs (SuperScript II RT, Invitrogen), which were then used as templates for quantitative real-time PCR [39 (link)]. The rp49 gene was used as an internal reference for normalizing the quality of total RNAs. Real-time PCR was performed with SYBR green using ABI7300 Real-time PCR Instrument (Applied Biosystems). Expressional fold of the various genes were determined by the comparative C_T method (ABI Prism 7700 Sequence Detection System User Bulletin #2, Applied Biosystems).

About 20 flies’ muscles and 80 hearts were homogenized in TRIzol. 10 μg of the total RNA was purified by organic solvent extraction from the TRIzol (TRIzol, Invitrogen). The purified RNA was treated with DNase I (RNase-free, Roche), and it was used to produce oligo dT-primed cDNAs (SuperScript II RT, Invitrogen), which were then used as templates for quantitative real-time PCR. The rp49 gene was used as an internal reference for normalizing the quantity of total RNAs. The real-time PCR was performed with SYBR green using an ABI7300 Real-time PCR Instrument (Applied Biosystems), with 3 biological replicates. Expression of the various genes was determined by the comparative CT method (ABI Prism 7700 Sequence Detection System User Bulletin #2, Applied Biosystems). Primer sequences of FOXO were as follows: F: 5′-AACAACAGCAGCATCAGCAG-3′; R: 5′-CTGAACC CGAGCATTCAGAT-3′. Primer sequences of PGC-1α were as follows: F: 5′-TGTTGCTGCTACTGCTGCTT-3′; R: 5′-GCCTC TGCATCACCTACACA-3′. Primer sequences of Mhc were as follows: F: 5′-TGCGTTGCCATCAATCCT-3′; R: 5′-GTAGGCAC CGTCAGAGATGG-3′. Primer sequences of Rp49 were as follows: F: 5 -CTAAGCTG TCGCACAAATGG-3′; R: 5′-AACTT CTTGAATCC GGTGGG-3′.