Thermal cycler dice tp800

Genomic DNA was eliminated from total RNA by TURBO DNA-free™ Kit (Thermo Fisher Scientific, Waltham, MA). First-strand cDNA was synthesized from 1 μg of total RNA using the SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific) with random hexamers. The PCR reaction mixture consisted of each primer pair (0.2 μM) and GoTaq® qPCR Master Mix (Promega, Madison, WI). Primer sequences and annealing temperature are shown in Supplemental Table S5. Data were analyzed using the Thermal Cycler Dice® TP800 (Takara, Shiga, Japan) and Thermal Cycler Dice® Real Time System Software Ver.5.11. Dissociation curve analysis was performed to ensure only a single peak was amplified. The expression levels of mRNA were normalized to EF1α in respective samples. Data were expressed Z-score and means ± SEM.

CD26-positive MM cell lines MSTO-clone12 and JMN were treated with unconjugated YS110 (40 µg/mL) or Y-TR1 (40 µg/mL) for 1 h before heat shock (45 °C, 2 h). After heat shock, the cells were lysed immediately to isolate the total RNA using an RNeasy mini kit (Cat. No. 74104, Qiagen, Hilden, Germany). Total RNA was reverse transcribed using Prime Script RT enzyme (Takara Bio Inc., Shiga, Japan), and cDNA was used for real-time PCR with the following primers. HSP70 (forward): CAC CAC CTA CTC CGA CAA CCA, HSP70 (reverse): GCG CCT AAT CTA CCT CCT CAA TG, (Invitrogen, Carlsbad, CA) beta actin (forward): TGG CAC CCA GCA CAA TGA A, beta actin (reverse): CTA AGT CAT AGT CCG CCT AGA AGC A (Takara Bio inc., Shiga, Japan). Real-time PCR reactions were performed using Thermal Cycler Dice TP800 (Takara Bio Inc.). The experiment was performed in triplicate, and the representative experiment is shown.

Wild-type, myc mutant, and MYC-overexpressing plants were grown on 1/2 MS agar plates at 23 °C for 10 d under a long-day photoperiod (16 h light/8 h dark). The plants were then subjected to cold treatments at 4 °C. Total RNA was isolated using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions^{16 (link)}. cDNA was synthesized from 2 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). The primers used for real-time PCR are listed in Table S2. Real-time PCR amplification and detection were carried out using SYBR Premix ExTaq (Takara Bio, Japan) on a Thermal Cycler Dice TP800 (Takara Bio, Japan). The relative transcript abundance was calculated using the comparative C_T method as previously described^{15 (link)}, with Actin2/7 as the control standard. Values representative the means ± SD (n = 3) from representative experiments from three or more independent experiments.

Screening for CNV in identified ar LCA-associated genes was performed for families in which the utilised TS or additional mutation screening approaches revealed only a single heterozygous rare variant (Supp. Table 3). SALASA MLPA probemix reagents (P221 for CRX, and P222 for RPGRIP1, GUCY2D, and CEP290; MRC Holland, Amsterdam, Netherland) were used to conduct an MLPA, as previously described⁴⁰ . A qPCR analysis was performed to confirm the family JIKEI-145 segregation analysis findings, using appropriate primers (available upon request), SYBR Premix EX Taq II (Takara, Japan) and a Thermal Cycler Dice TP800 (Takara, Japan), according to the manufacturer’s instructions. Relative comparative threshold cycle (Ct) values were calculated on the basis of the second derivative maximum method using dedicated software (Thermal Cycler Dice Real Time System software, v 5.11B for TP800, Takara, Japan). The relative copy number (RCN) was determined on the basis of the comparative ddCT method using the unaffected father DNA as a control (where an RCN score of 1.0 represented no copy number change, a score of 0.6–1.4 was considered abnormal, and a score <0.6 and >1.4 represented deletions and duplications, respectively). All reactions were performed in duplicate, and all experiments were repeated in triplicate.