Thermal cycler dice real time system tp 800 instrument

Total RNA was extracted from cells of each strain cultured in 20 ml of DPY medium for 7 days. cDNA was synthesized using SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo) according to the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) analysis was performed using Thunderbird SYBR qPCR Mix (Toyobo) and a Thermal Cycler Dice Real Time System TP-800 instrument (Takara) essentially as described previously^{48 (link)}. Each cDNA sample was analyzed in triplicate. The transcript level was analyzed using primers as follows (sequences are summarized in Supplementary Table 2): YHK188 and YHK189 for amyB (AO090120000196); YHK190 and YHK191 for glaA (AO090010000746); and YHK192 and YHK193 for pepA (AO090120000474). The expression level of each gene was normalized to that of actA (AO090701000065) using primers YHK194 and YHK195.

RNA was extracted using the High Pure RNA Isolation Kit (Roche, Tokyo, Japan), and cDNA was prepared as previously described [24 (link)]. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) and a Thermal Cycler Dice Real Time System TP-800 instrument (Takara, Shiga, Japan) as previously described. Samples were analyzed in triplicate, and SIRT3 expression levels were normalized to the corresponding ACTB (β-actin) levels. The PCR primer sequences used were as follows: SIRT3 forward primer 5′-CTGTACAGCAACCTCCAGCA-3′ and reverse primer 5′-CTCCTTGGCCAAAGTGAAAA-3′; ACTB forward primer 5′-TGGCACCCAGCACAATGAA-3′ and reverse primer 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′.

RNA was isolated using the High Pure RNA Isolation Kit (Roche, Mannheim, Germany). cDNA was synthesized using the ReverTra Ace Kit (Toyobo, Osaka, Japan). qRT-PCR was performed using the KAPA SYBR FAST qPCR Kit (KAPA Biosystems, Woburn, MA) and the Thermal Cycler Dice Real Time System TP-800 instrument (Takara, Shiga, Japan), as described27 (link). The samples were analyzed in triplicate, and gene expression levels were normalized to β-actin. The PCR primers are listed in Table S2.