Revertaid h minus first strand cdna synthesis kit

Retrotranscription to first strand cDNA was performed using RevertAid H Minus First Strand cDNA Synthesis kit (Themo Fisher Scientific ref: K1632). Briefly, 2000 ng of total RNA was used for cDNA synthesis following manufacturer’s instructions. 5 ng of original RNA was used to perform fast qPCR using GoTaq qPCR Master Mix (Promega ref: A6002) in a LigherCycler 480 (Roche) with the manufacturer’s protocol (Supp. Table 3). Primers were designed using OligoPerfect design (Thermo Fisher Scientific) and validated using in silico PCR (UCSC genome Browser) and Ensembl BLAST (Ensemble.org). Primers were purchased from Sigma-Aldrich (Supp. Table 4) and used at 0.5 μM final concentration. Finally, primer efficiency was tested using serial dilutions from a mixed pool of cDNAs from all the samples. All primers used showed an efficiency of 1.9 or above (Supp. Table 4).

The total RNA of the samples (the hydrated cysts, nauplii, juveniles and adults) was prepared using the RNeasy mini kit (Qiagen, Hilden, Germany). The total RNA was used to synthesize cDNA in a 20 μL reaction by using poly-T primer and RevertAid H Minus First Strand cDNA Synthesis Kit (Thermal Scientific, Waltham, MA, USA). The 20 μL of reaction mixture contained 4 μL of 5X reaction buffer, 2 μL of 10 μM dNTPs mix, 20 units of ribonuclease inhibitor, 200 units of RevertAid H Minus M-MuLV Reverse Transcriptase, 1 μL of oligo-dT primer and 500 ng of total RNA. Subsequently, the reaction mixture was incubated for 70 min at 42 °C. The reaction was terminated by heating at 70 °C for 5 min and then cooled to 4 °C. Complementary deoxyribonucleic acids (cDNA) were then used as a template in PCR for further steps.

NRK cells were transfected with scrambled or Dvl1 shRNA clones as described above using electroporation and Nucleofector (Lonza). After 24 h, cells were washed in cold PBS and homogenized using Trizol Reagent (Life Technologies). Total RNA was then extracted using Direct-zol columns (ZymoResearch). RNA concentration was quantified using a NanoDrop ND-100 (Thermo Scientific). Up to 2000 ng of RNA were used for cDNA synthesis using RevertAid H Minus First Strand cDNA Synthesis kit (Themo Fisher Scientific). Five nanogram of original RNA was used to perform fast qPCR using GoTaq qPCR Master Mix (Promega) in a LigherCycler^® 480 (Roche). Primers for Dvl1 and three housekeeping genes (Gapdh, Actb and Rps18) were designed using OligoPerfect design (Thermo Fisher Scientific) and validated using in silico PCR (UCSC genome Browser) and Ensembl BLAST¹. Primers (Dvl1 Fw: 5′-GCTGAAGCATGGTTTCCTGC-3′; Dvl1 Rv: 5′-GTTGAGGTTCAGGGATGCGA-3′; Actb Fw 5′-GGCTCCTAGCACCATGAAGA-3′; Actb Rv: 5′- CTGGAA GGTGGACAGTGAGG-3′; Gapdh Fw 5′-AGACAGCCGCATC TTCTTGT-3′; Gapdh Rv: 5′-CTTGCCGTGGGTAGAGTCAT-3′; Rps18 Fw 5′-CTTCCACAGGAGGCCTACAC-3′; Rps18 Rv: 5′-GTACTCGCAGGATGTGCTGA-3′) were used at 0.5 μM (Sigma Aldrich).

Mag Jet RNA Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for the extraction of total RNA. The RNA’s quality was estimated by electrophoresis in the presence of 1 μg/mL ethidium bromide (2% agarose gel in Tris/Borate/EDTA buffer). The concentration of the extracted RNA was determined with NanoDrop 1000c spectrophotometer. RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for reverse transcription of total RNA.

The pET11a-NfrGFP-Z, pMRBAD-Z-CfrGFP, pET11a-NfrGFP-BARD1, and pMRBAD-BRCA1-CfrGFP expression vectors were obtained and generated as previously described [14 (link)]. The BRCA2 fragments encoding the Helical Domain (HD, amino acids 2480–2667), three oligonucleotide-binding folds (OB1, amino acids 2668–2807, OB2, amino acids 2808–3048, OB3, amino acids 3050–3185), the full-length coding sequences of the DSS1, and the UbcH5a genes were obtained by PCR amplification of cDNA prepared from 293T cells using a RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA), and cloned into pET11a-NfrGFP-Z between XhoI and BamHI restriction sites (HD, OB1, OB2, OB3, DSS1, and UbcH5a), and pMRBAD-Z-CfrGFP between NcoI and AatII restriction sites (HD, OB1, OB2, OB3, and DSS1) after removing the leucine zipper motifs (Z). All of the BRCA1 and BRCA2 variants were obtained by direct mutagenesis of pMRBAD-BRCA1-CfrGFP and of pET11a-NfrGFP-BRCA2HD/OB1 using the QuickChange XL Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), according to the manufacturer’s instruction. DNA sequencing (Eurofins Genomics, Ebersberg, Germany) technology checked the recombinant clones.