Superscript vilo synthesis kit

DNA and RNA were extracted from embryo trunks (stage 09, n = 15 per temperature), AKG complexes (stage 15, n = 12 per temperature), and gonads (stage 22, n = 10 per temperature) using Qiagen AllPrep DNA/RNA ™ Mini (stages 9 and 15) and Micro (stage 22) kits, following the manufacturer’s instructions. Differences in the sample size per stage were due to embryonic mortality at later stages of development due to cadmium exposure. RNA and DNA were quantified using a NanoDrop Spectrophotometer. DNA was stored at −20 °C until further use.
The RNA extraction protocol from the extraction kits included a treatment with DNAse to reduce gDNA. RNA quality was assessed by the presence of ribosomal bands in 1% agarose gels. RNA was stored at −80 °C until 500 ng of RNA was converted to cDNA using Invitrogen SuperScript™ VILO™ Synthesis kit following the manufacturer’s instructions; cDNA was stored at −20 °C until further use.

RNA was extracted from trunks of stage 9 embryos when the gonadal primordium cannot be separated (n = 15 per T° per replicate), adrenal-kidney-gonad (AKG) complex of stage 12 embryos (when genital ridge may be present in C. picta given that urogenital tissue is present by this stage in T. scripta (Spotila, Spotila & Hall, 1998 ), from AKG of stage 15 embryos (n = 15 per T° per replicate) (when bipotential gonads could not be separated from AK), and from separated gonads from stage 19 (n = 13 per T° per replicate) and stage 22 (n = 12 per T° per replicate) embryos using Qiagen Rneasy™ Mini (stages 9, 12 and 15) and Micro (stages 19 and 22) kits, following the manufacturer’s instructions. RNA was quantified using a NanoDrop Spectrophotomoter and RNA quality was assessed by the presence of ribosomal bands in 1% agarose gels. Extracted RNA was stored at −80 °C until processing. All RNA was extracted from individual embryos and 200 ng to 1 µg of RNA was retro-transcribed to cDNA by RT-PCR using Invitrogen SuperScript™ VILO™ Synthesis kit following the manufacturer’s instructions. cDNA was stored at −20 °C.

The RNA of V. vermiformis CDC-19 was extracted using the RNeasy mini kit (Cat No: 74104, Qiagen). RNaseOUT (Thermo Fisher Scientific, San Jose, CA, USA) was added to the 50 μL volume of eluted RNA, thus preventing RNA degradation. To ensure of the absence of DNA contamination, two cycles of DNase treatment with 30 min of incubation at 37 °C were performed using TURBO DNase (Invitrogen, Carlsbad, CA, USA). Total RNA was purified using the RNeasy MinElute Cleanup Kit (Cat No: 74204, Qiagen) according to the manufacturer’s instructions. cDNA amplicons were obtained using the SuperScript VILO Synthesis Kit (Invitrogen) with random primers. The amplicons were purified using the Agencourt AMPure XP beads (Beckman Coulter, Inc.), then sequenced on the MiSeq instrument using the Nextera XT DNA sample prep kit (Illumina), with a paired-end strategy and a read length of 125 bp. The cDNA was visualized and quantified on a LabChip Bio-analyzer (Agilent Technologies). Fragmentation, tagging and barcoding were performed over 12 PCR amplification cycles. The library was purified on Agencourt AMPure XP beads (Beckman Coulter Inc.), normalized on specific beads, and pooled for sequencing.

RNA was isolated from HUVECs with Purelink® RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer's instructions. cDNA was synthesized with SuperScript® VILO™ synthesis Kit (Invitrogen, Carlsbad, CA). Quantitative RT-PCR was done using Roche SYBR-Green® master mix and a LightCycler 480 II Real-Time PCR system (Roche Applied Science, Penzberg, Germany). Crossing point (Cp) value was calculated and normalized to Cp values of housekeeping genes succinate dehydrogenase complex subunit A (SDHA) and beta-actin.

Each liver was homogenized and total RNA was isolated using TRIzol reagent (Invitrogen) and RNeasy mini kit (QIAGEN). First strand cDNA was synthesized using SuperScript VILO synthesis kit (Invitrogen). Quantitative RT-PCR (RT-qPCR) was performed using TaqMan gene expression probes in a LightCycler 480 real time PCR system (Roche). Primer information can be provided upon request.