Real-time quantitative PCR analysis of intestine of gilthead seabream
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ABSTRACT:
ORGANISM(S): Sparus aurata
PROVIDER: GSE289127 | GEO | 2026/02/07
REPOSITORIES: GEO
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Project description:Corneal stromal cells from six donors were used and expanded in standard conditions (FBS as a supplement) and xenofree conditions (HPL as a supplement). Bone marrow MSCs from one other donor were used as a comparative control. From the purified RNA, 10ng was used per sample to synthesize cDNA using the iScript Advanced cDNA synthesis kit (Bio-Rad Laboratories, Temse, Belgium) following the protocol supplied by the manufacturer. qRT-PCR assays were prepared in triplicate in 96-well PrimePCR plates for human MSCs (Bio-Rad Laboratories, Temse, Belgium), in which the primers come lyophilized per well. A total of 88 genes was targeted, which account for stemness, differentiation (osteo-, chondro-, adipo-, teno- and myogenesis), MSC-specific, and MSC-related genes. The thermal cycler used to perform the PCR was the CFX96 Real-Time System – C1000 Thermal Cycler (Bio-Rad Laboratories, Temse, Belgium). SsoAdvanced universal SYBR Green supermix was used to quantify double-stranded DNA. Dissociation of double-stranded DNA was assessed in melting curve analysis to determine the specificity. Three reference housekeeping genes were included in the assay plates (TBP, GAPDH and HPRT1) to normalize expression using the ΔΔCq method. The abundance of the transcripts in CS-MSC relative to the control BM-MSC was determined by calculating the relative normalized expression (RNE) or fold change. All analyses were performed using the Bio-Rad CFX Manager software. When gene expression exceeds a more than threefold change compared to BM-MSC it is deemed to be up- or downregulated.
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Whole-genome sequencing (WGS) was performed for 60 pairs of tumor-normal samples from patients diagnosed with NKTL. Genomic DNA from tumor tissue was extracted with QIAamp DNA Mini Kit. The DNA for the matching normal was obtained from blood or buccal swabs and purified by Blood and Cell Culture DNA Mini kit or E.Z.N.A. Tissue DNA Kit (Omega Bio-tek) according to manufacturer’s instructions. The quantity and quality were assessed by Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and agarose gel electrophoresis. All sequencing libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina). Paired-end sequencing was performed on Illumina HiSeq 2000.
Whole-transcriptome sequencing (WTS): Total RNA from snap frozen EITL tumor samples was extracted using TRIzol (Invitrogen) and purified with RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. The integrity of RNA was determined by electrophoresis using 2100 Bioanalyzer (Agilent Technologies). 500 ng of total RNA was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantification was performed using SsoFast EvaGreen Supermix and CFX96 Real-Time PCR System (both Bio-Rad). Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero (Illumina) and WTS was performed on Illumina HiSeq 2500 with 2x101 bp read length.
Description of prefix used in filenames:
T: Tumor samples
N: Normal samples (Blood)
P: PDX samples
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Project description:RNA was extracted from cells using Aurum Total RNA kit from Bio-Rad Laboratories, Inc., Hercules, CA following the manufacturerM-bM-^@M-^Ys recommendations. Gene expression profiling was performed using the BeadChip HumanHT-12 v4 Expression kit from IlluminaM-BM-., which contains 47,231 gene-probes (IlluminaM-BM-. Inc., San Diego, CA). The raw signal intensities were imported and analyzed using the GenomeStudioM-BM-. data software. After background subtraction and normalization, the signal intensity values were exported to the PartekM-BM-. genomics expression analysis suite using M-bM-^@M-^\Partek's Report Plug-inM-bM-^@M-^] option in the GenomeStudioM-BM-. software. Differentially expressed genes in the dox- versus vehicle-treated samples were identified using the M-bM-^@M-^\gene expressionM-bM-^@M-^] workflow in the PartekM-BM-. software. T47D cells hoaboring a doxycycline inducible shPIP construct were treated for 24 or 48 hours with 250 ng/mL of doxycycline and their mRNAs analyzed in biological triplicates (a total of 12 samples) using IlluminaM-bM-^@M-^Xs HumanHT-12 v4 BeadChips. The samples for each time point comprised of three samples each for doxycycline or equal volume of water as vehicle control.
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