Viiatm7 thermalcycler

Selected miRNAs were retro-transcribed with RT-TaqMan^® MicroRNA Assays: miR-125b, miR-200b, miR-225 (Applied Biosystems) on Veriti^® 96-Well Thermal Cycler (Applied Biosystems) under following conditions: 16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min. Subsequently, obtained cDNA were amplified using corresponding TaqMan^® MicroRNA Assays (Applied Biosystems) on ViiaTM7 Thermalcycler (Applied Biosystems) under following conditions: 50 °C for 2 min, 95 °C for 10 min and 95 °C for 15 s, 60 °C for 1 min followed by 40 cycles in D. rerio samples. Relative quantification was performed using the ΔΔCt method using U6 snRNA as housekeeping. Differential levels of each miRNA expressed were evaluated by ViiaTM7 software (Applied Biosystems).

cDNA was synthesized from 1 μg of total RNA. For reverse transcription, the QuantiTect^® Reverse transcription kit and gDNA wipeout (Qiagen, Hilden, Germany) were used according to the supplier’s protocol. Reactions were performed in Veriti^® 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). cDNA concentration was measured by means of NanoDrop UV/Vis micro-spectrophotometry (ND-1000; NanoDrop Technologies, Wilmington, DE, USA). cDNA samples were stored at −20 °C.
qPCR experiments were performed using 200 ng cDNA and Power SYBR™ Green PCR Master Mix (Thermo Fisher) and ViiaTM7 Thermalcycler (Applied Biosystems). Oligonucleotides to amplify D. rerio: AKT, Nrf2a, CBSb, CSE, GAPDH genes were ordered from Thermo Fisher, appropriate oligo sequences are indicated in Table S5. Amplification conditions were the following: 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, annealing step was carried out at 50 °C for 60 s and 72 °C for 30 s, to determine the relative quantities of the genes of interest transcripts present in the various experimental conditions compared with baseline. All our real-time PCR experiments were performed using GAPDH housekeeping gene as an internal control. We used the ΔΔCt method described in “Users bulletin”, ABI PRISM 7700 Sequence Detection System 1997. The data were handled by Real-Time PCR System ViiaTM7 software.

miRNA expression was determined using samples obtained from untreated control EA.hy926 cells and cells exposed with 0.3 μM lanthionine during 12–48 h. miRNA were retro-transcribed with RT-TaqMan^® MicroRNA Assays: miR-200c (RT/TM 002300), miR-423 (RT/TM 002340) (Applied Biosystems, Pleasanton, CA, USA) on Veriti^® 96-Well Thermal Cycler, under the following conditions: 16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min. Subsequently, cDNA were amplified using corresponding TaqMan^® MicroRNA Assays (4366596, Applied Biosystems, Vilnius, Lithuania) on a ViiaTM7 Thermalcycler under the following conditions: 50 °C for 2 min, 95 °C for 10 min and 95 °C for 15 s, 60 °C for 1 min followed by 40 cycles. Relative quantification was performed using the ΔΔCt method using U6 snRNA (RT/TM 0019732, Applied Biosystems, Pleasanton, CA, USA) as a housekeeping gene product. Differential levels of each miRNA expressed were evaluated by the ViiaTM7 software.