Thermal cycler tp800

The following experiments were performed using kits according to the manufacturer's instructions. Total RNA was isolated using ISOGEN (Nippon gene, Toyama, Japan). RT was performed using PrimeScript RT Master Mix (Takara, Shiga, Japan). cDNAs were quantified by real-time PCR using SYBR qPCR mix (Toyobo, Osaka, Japan) and a Thermal Cycler TP800 (Takara). Primer sets for qPCR are shown in Table S2.

Total RNA was isolated using ISOGEN reagent (Wako). Reverse transcription (RT) was performed using PrimeScript RT Master Mix (Takara, Shiga, Japan) according to the manufacturer’s instructions. cDNAs were quantified by real-time PCR using SYBR qPCR mix (Toyobo, Osaka, Japan) and a Thermal Cycler TP800 (Takara). RT primers were as follows: AR (s) 5′-ATGGTGAGCAGAGTGCCCTA-3′, (as) 5′-TCTGGGGTGGAAAGTAATAGTCAA-3′; CNPY2 (s) 5′- GACCATGCCCTGCACATATC-3′, (as) 5′- TAAAAGGCATTGCCACCATT-3′; GAPDH (s) 5′-GCACCGTCAAGGCTGAGAAC-3′, (as) 5′-TGGTGAAGACGCCAGTGGA-3′; KLK3 (s) 5′- TCTGCGGCGGTGTTCTG-3’, (as) 5′- GCCGACCCAGCAAGATCA-3′; TMPRSS2 (s) 5′- GGACAGTGTGCACCTCAAAGAC-3′, (as) 5′- TCCCACGAGGAAGGTCCC-3′; NKX3-1 (s) 5′- CCCAGTCCACTGAGCAAGCA-3′, (as) 5′- GGGACCCATTATAGGCAATAAACAC-3′.

Relative contents of type 1 inositol 1,4,5-tris phosphate receptor (IP3R1), type 1 ryanodine receptor (RyR1) and type 2 sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) were quantified by real-time PCR, according to the protocol in our previous study^{37 (link)}. Total RNA preparations from 5 mice in each genotype were used as templates for cDNA synthesis (ReverTra ACE qPCR-RT kit, TOYOBO, Japan). The mRNA content was analyzed using a real-time PCR system according to the manufacturer’s instructions (Thermal Cycler TP800, TaKaRa Bio, Japan). The cycle threshold (Ct) was determined from the cDNA amplification curve as an index for relative mRNA content in each reaction.

Lung samples were incubated in RNA stabilization reagent for 24 h at 4 °C and stored at −80 °C. Total RNA isolation was performed with an RNAeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription and complementary DNA (cDNA) synthesis were performed using an RT² First Strand Kit (Qiagen) and 1 μg RNA. Commercially available PCR arrays, including an Angiogenesis Array, were obtained from SA Biosciences (Frederick, MD). The PCR array contained 84 primer pairs that amplify genes involved in rat angiogenesis. Briefly, cDNA was mixed with 2 × RT² SYBR Green Master Mix (1.35 mL; Qiagen) and RNase-free water to a final volume of 2.7 mL. Each well in the RT² Profiler PCR array plate contained 25 μL of sample. PCR was performed on a Takara TP-800 thermal cycler (Takara, Shiga, Japan) following the manufacturer’s instructions. The housekeeping genes hypoxanthine phosphoribosyltransferase 1 (Hprt1) and ribosomal protein lateral stalk subunit P1 (Rplp1) were used to normalize expression levels, calculated using ΔCt values. Fold-changes in expression were calculated using the ΔΔCt (threshold cycle) method. ΔΔCt values in the EC and EC–ASC groups were determined and the fold-changes were calculated as 2^(−ΔΔCt).

Thyroid samples of 4W and 8M rats 24 h after 8 Gy irradiation and non-irradiated thyroid samples were incubated in RNA Stabilization Reagent for 24 h at 4°C and stored at −80°C. Total RNA was extracted using QIAzol Lysis Reagent and the RNeasy Mini Kit according to the manufacturer's instructions. cDNA synthesis and reverse transcription were performed using the RT² First Strand Kit and 1 μg of total RNA. cDNA (102 µl) was mixed with 1.35 ml of 2 × RT² SYBR Green Master Mix and RNase-free water to a final volume of 2.7 ml. Each well in the RT² Profiler PCR Array plate contained a 25-µl sample; samples were analyzed in triplicate. The PCR array contained 84 primer pairs that amplify genes involved in rat autophagy. Reactions were performed using a Takara TP-800 thermal cycler (Takara, Shiga, Japan) following the manufacturer's instructions. All kits and reagents and the PCR array (catalog no. PARN-084ZA) were purchased from Qiagen (Tokyo, Japan). Fold changes in expression were calculated using the ΔΔCt method (http://www.SABiosciences.com/pcrarraydataanalysis.php). The average expression of the most stable housekeeping gene (ACTB) in the array (the ‘normalization’ gene) was used to calculate ΔCt values. ΔΔCt values in the non-irradiated and irradiated groups were determined, and the fold change was calculated as 2^(−ΔΔCt).

Total RNA was extracted from frozen 1-cm root tips of 5-day-old pea seedlings using an RNAqueous column with Plant RNA Isolation Aid (Ambion, Austin, TX, USA). cDNA was synthesized from 1 µg total RNA with a QuantiTect reverse transcription kit (Qiagen, Hilden, Germany). Real-time qRT-PCR using SYBR Green I was carried out using a TP800 thermal cycler (Takara Bio, Shiga, Japan) as described previously (Sawada et al., 2008 (link)) using gene-specific primers. The mean expression level of two replicates was normalized to that of 18S rRNA as the internal control. Total RNA was extracted from Arabidopsis by the method of Suzuki et al. (2004) (link) using root tissues of plants grown hydroponically in modified MGRL medium for 1 week in the presence of 4 µM AlCl₃, as described previously (Kobayashi et al., 2007 (link)). Transcript levels of CYP51G1 and UBQ1 were quantified by real-time PCR using an Applied Biosystems 7300 instrument (Applied Biosystems, Foster City, CA, USA) and specific primer pairs, following the manufacturer’s instructions.

Total RNA was extracted from 1-cm root tips of cv. Rikuu-132 (R₁₃₂) and cv. Koshihikari (Ko) after 24 h treatment with 0.2 mM CaCl₂ with or without 10 µM AlCl₃ (pH 4.9) under L or DMG. Extraction and purification of the total RNA was performed using an RNAqueous column with Plant RNA Isolation Aid (Ambion, Austin, TX, USA). cDNA was synthesized from 1 µg total RNA with a QuantiTech reverse transcription kit (Qiagen, Hilden, Germany). Real-time qRT-PCR using SYBR Green I was carried out with a TP800 thermal cycler (Takara Bio, Shiga, Japan) as described previously (Wagatsuma et al., 2015 (link)) using the following gene-specific primers; 5′-GGACGTGGAAAGTCTGTGGT-3′ (sense) and 5′-AACAGCTGAACCAGCAAGGT-3′ (antisense) for OsHMG2, and 5′-AAGGCCTTCTTGGATTC-3′ (sense) and 5′-GCAGCAGCTGAATCTCATGT-3′ (anti-sense) for OsHMG3. The gene transcript levels were quantified by the standard curve method using a complementary DNA dilution series as described by Bustin et al. (2009) (link). The transcript levels of each gene were normalized to that of 18S rRNA.