Blue s green qpcr kit

To evaluate knockdown efficiency, RNA was isolated using the RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. 1 μg of RNA was reversely transcribed into cDNA using the cDNA Synthesis Kit (Biozym). Real-time RT-PCR was performed using the Blue S’Green qPCR Kit (Biozym) with three specific primers for PSMC1 (PSMC1_F1 5′-AAGAAAAGCAAGAGGAGGAAAG-3′; PSMC1-R1 5′-CACAGATGTAGACACGATGG-3′; PSMC1_F2 5′-AGCAAACCAAACCTCAGCC-3′; PSMC1_R2 5′-TCCCCTAGAATCAAATCCATCC-3′; PSMC1_F3 5′-ACACTACGTCAGCATTCTTTC-3′; PSMC1_R3 5′-ATCCATCAGCACCCCTATC-3′). Expression of PSMC1 was normalized to the expression of HPRT, which served as housekeeping gene (hHPRT-fwd 5′-TGGACAGGACTGAACGTCTTG-3′; hHPRT-rev 5′-CCAGCAGGTCAGCAAAGAATTTA-3′). Results are shown as 2^−ΔΔCt values.

RNA isolation from N. furzeri embryos and tissues using TRI reagent (Sigma-Aldrich) and quantitative Real Time RT-PCR analysis were performed as described (Zupkovitz et al., 2018b (link)). Total RNA was isolated from either snap-frozen whole embryos at different stages or brain, liver and muscle samples of adult fish following the manufacturer’s instructions. One microgram of isolated RNA was reverse transcribed with the iScript cDNA synthesis kit (Bio-Rad) and Real-time RT-PCRs were performed using the CFX384 Well qPCR Detection System (Bio-Rad) and Blue S’Green qPCR Kit (Biozym). TATA binding protein (tbp) housekeeping gene expression was used for normalization. All used primer sequences are listed in Supplementary Table 3. All expression raw values obtained from the Real-Time RT-PCR analysis (normalized to tbp) are listed in Supplementary Table 4. Real-time PCR experiments were initially analyzed with the CFX Maestro software. Graphical output and statistics were performed with Prism software (GraphPad). Statistical significances were calculated with one-way ANOVA implementing Tukey’s multiple comparisons. p-values were calculated employing Prism software and standard deviation (SD) is shown. p > 0.05; *: p ≤ 0.05; **p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.

For transcript level analysis, three clones per strain were cultivated in YPD medium (yeast extract 1%, peptone 2%, glucose 2%) in shake flasks overnight. Cells were washed and used to inoculate YNB medium (YNB without amino acids and ammonium sulfate 3.4 g L⁻¹, ammonium sulfate 10 g L⁻¹, potassium phosphate buffer 0.1 mol L⁻¹ pH 6, biotin 0.4 mg L⁻¹) with 2% glucose or xylose as carbon source. After 6 h of cultivation at 25 °C, cells were harvested and RNA was extracted according to the TRI reagent (Sigma-Aldrich) protocol, followed by DNase treatment with the Ambion DNA-free kit (Invitrogen) and cDNA synthesis with oligo(dT)23 primers (NEB) and the Biozym cDNA synthesis kit. RNA integrity was analyzed by agarose gel electrophoresis. Quantitative PCR was performed on a Rotor-Gene Q instrument (Qiagen) using the Blue S´Green qPCR kit (Biozym). Transcript levels were normalized to ACT1 expression and relative expression levels were calculated using the average expression of SOR1 in K. phaffii X-33 grown in YNB glucose as reference.

Quantitative RT-PCR was performed on four biological with two technical replicates each, probing homozygous H^WA and H^NN versus control H^cwt. Poly(A⁺) RNA was prepared from 12 third-instar wandering larvae with the PolyATtract^® System 1000 (Promega, Walldorf, Germany), followed by a DNase I (RNase free) digest (New England Biolabs, Ipswich, MA, USA). cDNA was synthesized from around 250 ng mRNA using the qScriber cDNA Synthesis Kit (HighQu, via Biozol, Eching, Germany). Real-time qPCR was performed as described before using the Blue S’Green qPCR Kit (Biozym, Scientific GmbH, Hessisch Oldendorf, Germany) on around 5 ng of cDNA and the MIC magnetic induction cycler (bms, via Biozym Scientific GmbH, Hessisch Oldendorf, Germany), including target and no-template controls [40 (link)]. Internal reference genes were cyp33 and Tbp. Primer pair sequences are listed at the DRSC FlyPrimer bank [50 (link)]: cnc (PP60393), cyp33 (PP14577), ewg (PP35080), GstD1 (PP16044), mTFB2 (PP26980) and Tbp (PP1556). Respective oligonucleotides were obtained from Microsynth (Balgach, Switzerland). The micPCR^® software version 12.2 was used for relative quantification of the data, based on REST and taking target efficiency into account [51 (link)].

For isolation of total RNA, samples were thawed and homogenized using a FastPrep 120 (Thermo Fisher Savant) and processed further using a Direct-zol RNA miniprep kit (Zymo Research). Total RNA was quantified using a BioSpectrometer (Eppendorf), and 2 μg of total RNA was used for reverse transcription in a 20-μl reaction mixture with random hexameric primers following the instructions of the RevertAid H minus first-strand cDNA synthesis kit (Thermo Fisher). Reverse transcription reaction mixtures were diluted 12-fold with nuclease-free water, and 2 μl of the dilution was used as the input for a 10-μl SYBR-based qPCR (Blue S′Green qPCR kit; Biozym Scientific) on an AriaMx real-time PCR system (Agilent). Primers are listed in Table S1E. For each sample, technical duplicates for each target gene were measured. Raw threshold cycle (C_T) data were exported to MS Excel and the Δ(C_T) values from technical replicates (shake flasks and reaction replicates) were averaged before calculating the ΔΔ(C_T) values according to the method in reference 76 (link), assuming an amplification efficiency of one. Actin (An15g00560) was used as the reference gene.

Total RNA was isolated from cells and tissues using peqGOLD RNAPure™ (VWR International, 30-1010) and innuSOLV RNA Reagent (analytikjena, Jena, Germany) according to the manufacturer’s instructions. A Precellys^® 24 (Bertin, Montigny-le-Bretonneux, France) was used (6000 rpm, 30 s) to homogenize the adipose tissue samples. Reverse transcription was performed using a Proto-Script^® First Strand cDNA Synthesis Kit (E6300L, New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. For qPCR analyses, gene-specific and intron-spanning primers were designed using Primer 3 software and were acquired from Microsynth (Balgach, Switzerland) unless otherwise noted (Table S1). A Biozym Blue S’Green qPCR Kit (331416XL, Biozym, Hessisch Oldendorf, Germany) and a Rotor-Gene Q (Qiagen, Hilden, Germany) were used according to the manufacturer’s instructions. Expression levels were quantified in duplicate and were calculated using internal amplification standards. 14-3-3 Protein zeta/delta (Ywhaz) was used as a reference gene for all samples for normalization.
Knockout of Smo was confirmed by assessment of Smo mRNA in liver tissue or hepatocytes. Only Hh-KO samples with Smo expression of less than 50% were included.