Whole-genome expressional analyses were carried out using Human Ref-8v3 or Human HT-12 v4 BeadArrays (Illumina, San Diego, CA). Total RNA was linearly amplified with biotin incorporation using Illumina TotalPrep kits (Life Technologies, Carlsbad, CA). The quality of the cRNA was assayed using an Agilent 2100 bioanalyzer (Santa Clara, CA). The resulting cRNA was hybrized to Illumina BeadChips (San Diego, CA), processed and read using a BeadStation array reader (Illumina, San Diego, CA) according to the manufacturer’s instructions. Numerical values of relative fluorescence units (RFU) were determined for indicated genes. Values less than 180 units were considered to be background signals. The RFU values for the two donors were averaged and these values were used to determine fold changes with respect to day 0 hMSCs. Table 1A -C and supplemental Tables present fold difference at the respective time points.
Totalprep kit
Manufactured by Illumina
Sourced in United States
The TotalPrep Kit is a laboratory equipment product designed for sample preparation. It provides a comprehensive solution for extracting and purifying nucleic acids from various sample types. The kit includes reagents and consumables necessary for the sample preparation process, enabling users to efficiently obtain high-quality nucleic acid samples for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Human ht 12 v4 beadarrays, by Illumina (2 mentions)
Taqman fast universal master mix, by Thermo Fisher Scientific (2 mentions)
Beadstudio 3, by Illumina (2 mentions)
High capacity cdna transcription kit, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Beadstation array reader, by Illumina (1 mentions)
Humanref 8 v3, by Illumina (1 mentions)
Iscan microarray scanner, by Illumina (1 mentions)
Beadchips, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Humanht 12 v3 beadchip, by Illumina (1 mentions)
Beadstudio software, by Illumina (1 mentions)
Beadstation 500g, by Illumina (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Human ht 12 v3 beadchips, by Illumina (1 mentions)
6 protocols using totalprep kit
1
Whole-Genome Expression Analysis of hMSCs
2
Genome-wide expression profiling of cells
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Total RNA was extracted from cells using Qiagen RNeasy mini kits according to instructions supplied by the manufacturer. RNA concentrations were measured using a Nanodrop spectrophotometer, and RNA integrity was determined by denaturing agarose gel electrophoresis or by an Agilent 2100 bioanalyzer. Whole-genome expression was obtained using Illumina Human HT-12 v4 BeadArrays. In preparation for Illumina BeadArrays, total RNA was linearly amplified and biotin-labeled using Illumina TotalPrep kits (Life Technologies, Temecula, CA, USA). The cRNA quality was measured using an Agilent 2100 Bioanalyzer before being hybridized to Illumina BeadChips, processed, and read by an iScan microarray scanner according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Values under 130 relative fluorescence units (RFUs) were considered as nonspecific background signal.
3
RNA Isolation and Real-Time PCR Analysis
4
RNA Extraction and Illumina Microarray
5
Chondrocyte Transcriptome Dynamics Assay
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Freshly isolated chondrocytes were allowed to adhere for 16 hours, and then their media were supplemented with 1 μM actinomycin D (Sigma) for 0, 1, 3, or 5 hours. At each time point, the cell layer was extracted using TRIzol, and total RNA was purified using chloroform-based phase separation and subsequent precipitation with isopropanol. RNA pellets were dissolved in RNase-free H2O and stored at −80°C until required. Total RNA from the 0-, 1-, 3-, and 5-hour actinomycin D time points from 4 samples from each group were used to probe Illumina HT-12 BeadChip arrays at The Genome Centre core facility at Queen Mary University of London. RNA integrity was confirmed using an Agilent bioanalyzer, and RNA was then labeled with Cy3 using an Ambion TotalPrep kit before being hybridized to arrays using an Illumina BeadStation 500G. Non-normalized data were exported from Illumina BeadStudio software and used for subsequent analysis.
6
RNA Expression Analysis by qPCR and Microarray
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RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN) and was reverse-transcribed with a High Capacity cDNA Transcription Kit (Life Technologies). Real-time PCR was performed using fluorogenic probe-primer combinations (Supplementary Table 7 ) specific for the target gene and TaqMan FAST Universal master mix (Life Technologies). All mRNA levels were normalized to levels of the control gene mouse Gapdh mRNA, determined with the TaqMan Rodent GAPDH Control Kit or human GAPDH mRNA with primers for GAPDH nucleotides 37–910 or OAZ1 mRNA with the IDT PrimeTime Pre-designed assay Hs.PT.42.328511.g from Integrated DNA Technologies47 (link).
Oligonucleotide microarray analysis was performed as described48 (link). Gene-expression analysis was performed using Illumina Mouse-WG6 v1.1 BeadChips for mouse samples, and Illumina HumanHT-12 v3 BeadChips for human samples (Illumina). Isolated total RNA was amplified and biotinylated with an Ambion Illumina TotalPrep Kit. Hybridization and scanning of BeadChip arrays was performed according to the manufacturer's instructions, with BeadStudio 3.0 software (Illumina). Treatment and conditions were randomized across BeadChip slides to avoid confounding. For bead-level analysis, raw data were exported from BeadStudio.
Oligonucleotide microarray analysis was performed as described48 (link). Gene-expression analysis was performed using Illumina Mouse-WG6 v1.1 BeadChips for mouse samples, and Illumina HumanHT-12 v3 BeadChips for human samples (Illumina). Isolated total RNA was amplified and biotinylated with an Ambion Illumina TotalPrep Kit. Hybridization and scanning of BeadChip arrays was performed according to the manufacturer's instructions, with BeadStudio 3.0 software (Illumina). Treatment and conditions were randomized across BeadChip slides to avoid confounding. For bead-level analysis, raw data were exported from BeadStudio.
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