Gdna wipeout

Total RNA was extracted from cell lysates using QIAGEN RNeasy Mini kits, following the manufacturer’s instructions. Complementary DNA was prepared from 300 ng of total RNA using QIAGEN reverse transcriptase kit, following elimination of genomic DNA using QIAGEN gDNA WipeOut. Since there is no discrepancy between protein level and mRNA expression of Angiopoietin-like 4 (ANGPTL4), only the gene expression was determined [9 (link)]. Gene expression was determined by real-time PCR using Eva Green Master Mix (Montreal Biotech) on a Rotor-Gene. Quantitect primers (forward and reverse) for ANGPTL4, metallothionein-3 (MT3), and β-actin were purchased from QIAGEN, with β-actin serving as the reference gene. Delta-delta CT (cycle threshold) analyses were conducted using the Rotor-Gene 6000 software version 1.7.

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.