Revertra acetm qpcr rt master mix with gdna remover

Total RNA was isolated from PDAC cells using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In total, 0.1 μg of total RNA was reverse transcribed using ReverTra Ace^TM qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka Japan) according to the manufacturer’s instructions. Finally, 500-fold diluted cDNA was amplified using LightCycler^® 480 SYBR Green I Master (Roche, Basel, Switzerland) and primer sets (Supplementary Table S1), and the amplicon was detected and analyzed by LightCycler480 System II (Roche, Basel, Switzerland).

Total RNA was isolated from the organs using TRIzol™ Reagent catalog number 15596026 (Invitrogen, Massachusetts, US). DNA synthesis was performed using ReverTra AceTM qPCR RT Master Mix with gDNA Remover product number FSQ-301 (Toyobo, Osaka, Japan) according to the manufacturer's protocol. Quantitative real-time RT-PCR was performed using the SensiFAST™ SYBR
^® No-ROX Kit product number BIO-98020 (Bioline, United Kingdom) according to the manufacturer's instructions and used Rotor-Gene Q quantitative real-time PCR machine (Qiagen, USA). The first step of Quantitative real-time RT-PCR was polymerase activation for one cycle at 95 °C for two minutes, then denaturation prosses at 95 °C for five seconds and annealing at 60 °C for 30 seconds. The denaturation and annealing process took 40 cycles. The PCR was performed using mGAPDH as a housekeeping gene and the primers from Integrated DNA Technologies, USA. The gene-specific primers for the cDNA used in this study are listed in
Table 1.

Four fish that were randomly selected in the previous section were used for the tissue extraction. Approximately 25 mg samples were harvested from the following 12 organs: brain (BR), gills (GL), heart (HR), liver (LV), spleen (SP), head kidney (HK), trunk kidney (TK), intestine (INT), stomach (SM), muscle (MC), skin (SK), and whole blood (WB). Whole blood was obtained via withdrawal from the caudal vein at caudal peduncle areas using a 1 mL syringe equipped with a heparinized 23-G needle. The total RNA was extracted from these 12 tissues (25 mg) using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. The precipitated RNA pellets from the target tissues were dried in air, and sterile nuclease-free water was used to dissolve the isolated total RNA. The quantity and quality of the obtained RNA were identified based on an absorbance ratio of OD260/280 nm using NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 1 µg of total RNA from each tissue was separately used to synthesize the 1st strand cDNA using ReverTra Ace^TM qPCR RT Master Mix with gDNA Remover (TOYOBO Bio-Technology, CO., LTD., Osaka, Japan). The 1st strand cDNA from all the tissues was synthesized based on the protocol recommended by the company and immediately stored at −20 °C in a deep freezer.

TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) was used to extract total RNA from H9c2 cells 24 h after treatment. Then, 1 µg of total RNA was reverse-transcribed using ReverTra AceTM qPCR RTMaster Mix with gDNA Remover (FSQ-301; Toyobo, Japan) to obtain complementary DNA. RNA levels of ANP and NFATC3 in H9c2 cells were quantified using SYBR Green PCR Master Mix Kit (QPS-201; Toyobo, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as an in-house control. A quantitative reverse transcription polymerase chain reaction (RT-qPCR) was conducted using a CFX96TM Real-Time System (Bio-Rad Laboratories, CA, USA) for 60 cycles. The sequences of primers for amplification were: ANP, 5′-GGTAGGATTGACAGGATTGGA-3′ and 5′-GCAGATTTGGCTGTTATCTTCG-3′; NFATC3, 5′-GGAAATCCCACTTCTACCTGAA-3′ and 5′-GCCAATATCAGTTTCTCCTTTTCG-3′, and GAPDH, 5′-AACCCATCACCATCTTCCAG-3′ and 5′-CCAGTAGACTCCACGACATAC-3′. Cycle threshold (Ct) values were used to measure the amount of amplified PCR product relative to GAPDH as the control.

Total RNA was extracted from the thalli and antheridial receptacles using ISOSPIN Plant RNA (NIPPON GENE). Next, total RNA (500 ng) was reverse-transcribed using ReverTra Ace^TM qPCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative real-time PCR was performed using THUNDERBIRDTM SYBR qPCR Mix (TOYOBO) in the StepOnePlus Real-Time PCR System (Applied Biosystems). The PCR cycling conditions were 95 °C for 15 min, 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. The gene expression level was normalized to that of MpACT1 to obtain a relative expression level.
The biological samples were analyzed in triplicate. Primers used for qPCR are listed in Supplementary Table 2.