Genome-wide gene expression profiling was conducted using MouseRef-8 v2.0 Expression BeadChips (Illumina, San Diego, CA), which contain ∼25,600 well-annotated RefSeq transcript features. Total RNA integrity was determined using the Experion RNA HighSens Analysis Chip (Bio-Rad, Hercules, CA) on the Experion Automated Electrophoresis System. RNA quality indicator values were >9.0 for all samples. Total RNA was labeled using TotalPrep RNA Labeling Kit (Ambion) before incubation on MouseRef-8 v2.0 BeadChips and imaging by the HiScan Array Scanner (Illumina, San Diego, CA) at the University of Chicago Genomics Facility (Chicago, IL). Four experimental replicates of aNSCs transduced with shKdm5b or control shScr lentiviral vector were simultaneously profiled on a single MouseRef-8 BeadChip. Raw data produced from HiScan imaging were outputted with the GenomeStudio software package and GEX module (Illumina).
Totalprep rna labeling kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TotalPrep RNA Labeling Kit is a laboratory equipment product designed for the preparation and labeling of RNA samples for microarray analysis. The kit provides the necessary reagents and protocols to convert RNA samples into biotinylated cRNA, which can then be hybridized to microarray platforms.
Automatically generated - may contain errors
Lab products found in correlation
Mouseref 8 v2.0 expression beadchip, by Illumina (2 mentions)
Biotinylated utp, by Thermo Fisher Scientific (2 mentions)
Humanht 12 v4 expression beadchip, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Genomestudio software package, by Illumina (1 mentions)
Mouseref 8 v2.0 beadchips, by Illumina (1 mentions)
Hiscan array scanner, by Illumina (1 mentions)
Mousewg 6 v2 expression beadchip, by Illumina (1 mentions)
Genomestudio software, by Illumina (1 mentions)
High pure rna isolation kit, by Roche (1 mentions)
Rnalater reagent, by Qiagen (1 mentions)
Experion automated electrophoresis station, by Bio-Rad (1 mentions)
Beadarray reader, by Illumina (1 mentions)
Ratref 12 expression beadchip, by Illumina (1 mentions)
Iscan system, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Tempus spin rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
8 protocols using totalprep rna labeling kit
1
Genome-wide Gene Expression Profiling of aNSCs
2
Illumina Mouse Gene Expression Profiling
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The Mouse WG-6 v2 Expression BeadChip (Illumina, San Diego, CA, USA) was processed in accordance with the manufacturer's instructions. Two hundred nanograms of total RNA was used for cRNA in vitro transcription and labeling with the TotalPrep™ RNA Labeling Kit using Biotinylated-UTP (Ambion, Austin, TX, USA). Hybridization is carried out in accordance with the Illumina Hybridization System Manual. The raw and background subtracted matrices extracted from Illumina Beadstudio are subject to a modified quantile normalization procedure where in the quantile distribution is estimated from the project samples, and negative values in the quantile distribution are scaled to fall within the range of 1 and 2. The resulting quantile distributions are then log transformed and then used to normalize each individual array following the typical quantile normalization procedure.
3
Transcriptome Analysis of RNA Samples
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Total RNA was prepared with the High Pure RNA Isolation Kit (Roche), and transcription profiles were generated using HumanHT-12 v4 Expression BeadChip (Illumina) by Health GeneTech Corp. services. The quality of each RNA sample was assessed using an Aglilent 2100 Bioanalyzer and NanoDrop-1000. RNA was amplified and labeled using the Ambion Illumina TotalPrep RNA Amplification kit (Invitrogen) and the TotalPrep RNA Labeling Kit (Ambion). The samples were hybridized for 16 h at 58°C to a HumanHT-12 v4 Expression BeadChip. After hybridization, the chip was washed, stained, and scanned with an iScan scanner. Microarray data were analyzed using GenomeStudio software version 2011.1 (Illumina) and normalized by Gene Expression Module software version 1.9.0 (Illumina). The hierarchical clustering and heatmap of differentially expressed genes were generated using Cluster 3.0 software and Java TreeView software version 1.1.4r4. After clustering, the functional annotations of transcripts were determined using DAVID Bioinformatics Resources v6.7 [61 (link)–62 (link)].
4
Transcriptome Analysis of Spleen Tissue
5
Illumina-based Kidney Transcriptome Analysis
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The collected samples were sent to JSC Genoanalytica (Moscow, Russia), where total RNA was extracted and processed. Three samples from ISIAH kidney and three samples from WAG kidney were run as experimental replicates. Four hundred nanograms of total RNA was used for complementary RNA in vitro transcription, followed by a T7 RNA polymerase-based linear amplification and labeling with the TotalPrep RNA Labeling Kit using Biotinylated-UTP (Ambion, Austin, TX). The signal was developed by staining with Cy3-streptavidin. The hybridization was performed on Illumina RatRef-12 Expression BeadChip microarray platform containing 22,524 probes for a total of 22, 228 rat genes selected primarily from the National Center for Biotechnology Information RefSeq database (Release 16; Illumina, San Diego, CA, USA). Hybridization, washing and staining were carried out according to the Illumina Gene Expression Direct Hybridization Manual. The BeadChip was scanned on a high-resolution Illumina BeadArray reader.
6
Transcriptome Analysis of PEO Patients
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Total muscle RNA was extracted with standard TRIzol and chloroform method and purified with RNA purification kit (RNeasy; Qiagen). A total of 250 ng of total RNA from four PEO patients and eight controls was used for global gene expression analysis on the HumanHT‐12 v4 Expression BeadChip (Illumina) with TotalPrep RNA Labeling Kit (Ambion). Chipster v2.1.0 software was used for quantile normalization (http://chipster.github.io/chipster/ ). Data were further filtered by coefficient of variation (standard deviation/mean; 50% of all present genes were filtered out). To determine the differently expressed genes between PEO or controls on ND and after mAD, paired empirical Bayes statistical test was applied together with the Benjamini–Hochberg P‐value adjustment method. For further analysis, the transcripts with adjusted P‐value < 0.05 for differential expression between PEO on standard diet and PEO on Atkins diet were selected regardless of fold change and subjected to pathway analysis using Ingenuity Pathway Analysis (IPA; Ingenuity® Systems; www.ingenuity.com ). All genes with log2 fold change > 0.26 and < −0.26 regardless of adjusted P‐value between controls on standard diet and controls on Atkins diet were used for IPA.
7
Genome-wide Transcriptome Profiling of DRGs
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Genome-wide RNA expression analysis was performed as previously described [24 (link), 25 (link)]. Briefly, RNA was isolated from DRGs with the RNeasy Mini Kit (Qiagen). Total RNA integrity was determined with the RNA Nano 6000 chip on the Agilent 2100 Bioanalyzer. Total RNA with a RNA integrity number (RIN) > 8.0 was labeled using a TotalPrep RNA Labeling Kit (Ambion) before hybridization on Rat Ref-12 Expression BeadChips (Illumina) and imaging by the Illumina iScan System at the Lerner Research Institute Genomics Core, Cleveland Clinic, Cleveland, OH. Rat Ref-12 BeadChips allow for probing of ~22,523 well-established and provisional annotated transcripts. Raw data generated from iScan imaging was outputted using the GenomeStudio software package and Gene Expression module.
8
RNA Extraction and Illumina Microarray
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Peripheral blood, 3 mL, was collected directly into Tempus RNA tubes, and frozen and stored at −20°C. Frozen samples from Mexico City and Quezon were batched and sent to the Feinstein Institute for Medical Research for RNA extraction and processing. RNA was extracted with the Tempus Spin RNA Isolation Kit (Ambion) according to the manufacturer's instructions and assessed for integrity and quantity using the Bioanalyzer (Agilent) and NanoDrop (NanoDrop Technologies). Total RNA, 50–200 ngs, was processed using the TotalPrep RNA Labeling Kit (Ambion) that has been optimised for use with Illumina's whole-genome expression platform. The RNA amplification process uses a streamlined protocol developed in the laboratory of James Eberwine.13 (link) The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter using a reverse transcriptase (ArrayScript), that catalyses the synthesis of virtually full-length cDNA. The cDNA then undergoes second strand synthesis and a clean-up step and is used as a template for in vitro transcription (IVT) (MEGAscript) with T7 RNA polymerase. Biotinylated-UTP is used in the IVT step to generate hundreds to thousands of biotinylated antisense RNA copies of each mRNA in a sample. The cRNA is subjected to a clean-up step, quantitated, labelled, hybridised to an Illumina microarray chip, stained and scanned.
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