Qubit rna br kit

To quantify abundance of RNA of targeted genes in stable mutant lines, 5-dpf larvae from a cross of heterozygous mutants were quickly euthanized in a Petri dish on dry ice, and a small piece of caudal fin tissue was carefully removed from each larva for DNA extraction and genotyping via high-resolution melt curves and SDS polyacrylamide gels, as previously described (primers listed in Supplementary Table S1). The larvae were individually placed in RNAlater (Thermo Fisher Scientific, Waltham, MA, United States), and RNA extraction was performed using the RNAeasy kit (Qiagen) with gDNA eliminator columns for DNA removal. RNA concentration from all samples was obtained with the Qubit RNA BR kit (Thermo Fisher Scientific, Waltham, MA, United States). RNA levels of syngap1b or slc7a5 in stable mutant lines were evaluated using a quantitative PCR with the Luna Universal One-Step RT-qPCR kit (New England BioLabs) following the manufacturer’s protocol. Fold change values were obtained using the ΔΔCq method, with raw Cq values normalized with expression of housekeeping gene β-actin (primers listed in Supplementary Table S1) and represented as fold changes to the average of the WT samples.

The quality and quantity of the extracted RNA samples were analyzed with a LabChip GX Touch HT RNA Assay Reagent Kit (PerkinElmer, Waltham, MA, USA) and Qubit RNA BR kit (Thermo Fisher Scientific, Waltham, MA, USA). For genomic DNA contamination measurement, a Qubit DNA BR kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. Dual-indexed mRNA libraries were prepared from 150 ng of total RNA with a QuantSeq 3′ mRNA–Seq Library Prep Kit FWD (Lexogen Gmbh, Vienna, Austria) according to user guide version 015UG009V0251. During second strand synthesis, 6 bp Unique Molecular Identifiers (UMIs) were introduced with the UMI Second Strand Synthesis Module (Lexogen Gmbh, Vienna, Austria) for detection and removal of PCR duplicates. The quality of the libraries was measured with a LabChip GX Touch HT DNA High Sensitivity Reagent Kit (PerkinElmer, Waltham, MA, USA). Sequencing was performed with a NovaSeq 6000 System (Illumina, San Diego, CA, USA) with read length of 2 × 101 bp and target coverage of 10 M reads for each library. QuantSeq 3′ mRNA–Seq Integrated Data Analysis Pipeline version 2.3.1 FWD UMI (Lexogen Gmbh, Vienna, Austria) on Bluebee^® Genomics Platform was used for primary quality evaluation of the RNA sequencing data.

Ten AGCT FFPE tissue sections of 5-µm thickness were mounted on objective slides, and areas with >80% AGCT cellularity (based on hematoxylin and eosin slide review) were scraped off into a microcentrifuge tube. Total RNA was extracted from the FFPE tissue using Maxwell RSC RNA FFPE kit (Promega, Madison, WI, USA), according to instructions. Quality and quantity of the extracted RNA samples were assessed with a 2100 Bioanalyzer using RNA 6000 Pico Kit (Agilent, Santa Clara, CA, USA) and Qubit RNA BR kit (Thermo Fisher Scientific, Waltham, MA, USA). For genomic DNA contamination measurement, a Qubit DNA BR kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. In order to avoid the batch effect, all RNA samples were extracted using the same automated extraction system by one researcher. Furthermore, all samples were sequenced on the same run.

Total RNA was extracted from approximately 0.5–1.5 g of tissue using a modified CTAB method [36 (link)], and stored at -80°C in 10mM Tris-HCl buffer (pH 8.0). Approximately 25 μg of total RNA from each sample was treated with DNase I enzyme to remove contaminating genomic DNA (gDNA) prior to confirmation of RNA integrity and gDNA removal by agarose gel electrophoresis. Absence of contaminants was confirmed spectrophotometrically using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, USA) and sample concentrations estimated using a Qubit RNA BR kit on the Qubit v1 fluorometer (Thermo Scientific).

Representative areas of tumor or normal tissue were marked on HE slides by a pathologist. Depending on the marking, DNA was extracted from the whole FFPE tissue sections or from six representative 0.8 mm cores with the ReliaPrep FFPE gDNA Miniprep System (Promega, Madison, WI, USA). DNA from FFPE samples that were selected for whole-exome (WES) and whole-genome sequencing (WGS) were extracted using the standard phenol-chloroform method. Fresh frozen tissue samples had been extracted with the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA, USA).
Total RNA was extracted and purified from macrodissected sections using the RNeasy® FFPE Kit (QIAGEN, Hilden, Germany) and the deparaffinization solution (QIAGEN) according to the manufacturer’s instructions. The concentration and purity of the extracted RNA were examined using the LabChip GX Touch HT RNA Assay Reagent Kit (PerkinElmer, Waltham, MA, USA) and the Qubit RNA BR kit (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA contamination was measured with the Qubit DNA BR kit (Thermo Fisher Scientific).

MCF7 and CAMA-1 cells were seeded into 6-well microplates in phenol red-free RPMI-1640 medium (Invitrogen) containing 2 mmol/L l-glutamine and 5% (volume/volume, v/v) charcoal-stripped FBS (Sigma). Cells were treated with 1 nmol/L estradiol and 100 nmol/L fulvestrant, AZD9496, or camizestrant for 24 hours, with three replicates per condition and non–estradiol-treated controls. Samples were harvested in 1 mL of QIAzol lysis buffer and snap-frozen. Snap-frozen tissue from PDX studies was homogenized and resuspended in QIAzol lysis buffer. RNA was extracted using RNeasy 96 QIAcube HT total RNA Cell with DNAse treatment, and samples randomized over 96-well plates. RNA was quantified using the Qubit RNA BR kit (Q10213 Invitrogen), per the manufacturer's instructions.

Slices of 10 µm were mounted on glass slides and left to dry overnight at room temperature. Macrodissection of tumour tissue was performed to obtain pure tumour tissue cleared of the normal adrenal cortex. Total RNA was extracted using the RNeasy FFPE Kit (Qiagen) as recommended by the manufacturer, and RNA concentration was measured with Qubit RNA BR kit (Invitrogen). RNA quality was measured using Bioanalyzer, and the DV200 values were calculated to be >85% for phaeochromocytoma samples and the negative control and 39% for the positive control. Thus, all samples meet the standard 30% cut-off.