Nanodrop uv vis micro spectrophotometry nd 1000

mRNA and miRNA were extracted from cells using mirVana™ PARIS™ kit (AM1556, ThermoFisher Scientific, Milan, Italy) according to the supplier’s protocol. RNA concentration was measured by means of NanoDrop UV/Vis micro-spectrophotometry (ND-1000; NanoDrop Technologies, Wilmington, DE, USA). Samples were stored at −80 °C.

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.

The mRNA was extracted from cells using a mirVana™ PARIS™ kit (AM1556, ThermoFisher Scientific, Milan, Italy) according to the supplier’s protocol. The RNA concentration was measured by means of NanoDrop UV/Vis microspectrophotometry (ND-1000; NanoDrop Technologies, Wilmington, DE, USA). The samples were stored at −80 °C.