The RNA isolation was performed using the RNeasy Mini Kit (Qiagen, USA) in accordance with the manufacturer’s instructions. A GeneChip protocol was used and the experimental procedures were performed in accordance with the Affymetrix GeneChip Expression Analysis Technical Manual. Using 5 µg of total RNA as template, a double-stranded DNA was synthesized by means of the One-cycle cDNA synthesis kit (Affymetrix, USA) and T7-(dT)24 primer. GeneChip Sample Cleanup Module (Qiagen, USA) was used to purify the DNA. By using an in vitro transcription (IVT) labelling kit (Affymetrix, USA) according to the manufacturer’s instructions, the IVT was performed to produce biotin labelled complementary RNA. Biotinylated complementary RNA was cleaned using the GeneChip Sample Cleanup Module (Qiagen), fragmented to 35–200 nucleotides, and hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays that contain ~55000 human transcripts. After washing, the array was stained with streptavidin–phycoerythrin (Molecular Probes). The staining signal was amplified by biotinylated anti-streptavidin (Vector Laboratories) followed by second staining with streptavidin-phycoerythrin, and then scanned on the GeneChip Scanner 3000.
Genechip sample cleanup module
Manufactured by Qiagen
Sourced in United States
The GeneChip Sample Cleanup Module is a lab equipment product that facilitates the purification and cleanup of RNA samples prior to downstream processing and analysis. It provides a streamlined workflow for removing contaminants and impurities from RNA samples to ensure optimal performance in subsequent applications.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
Genechip rat gene 2.0 st array, by Thermo Fisher Scientific (2 mentions)
Superscript system, by Thermo Fisher Scientific (2 mentions)
Streptavidin phycoerythrin, by Merck Group (2 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (2 mentions)
Genechip sample cleanup module, by Thermo Fisher Scientific (1 mentions)
Genechip ivt labeling kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Biotinylated anti streptavidin, by Vector Laboratories (1 mentions)
One cycle cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Human genome u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Genechip expression analysis, by Thermo Fisher Scientific (1 mentions)
Streptavidin phycoerythrin, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Fluidics station 400, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
4 protocols using genechip sample cleanup module
1
Affymetrix GeneChip Expression Analysis
2
Transcriptomic Analysis of Fenretinide Treatment
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Total RNA was extracted from cells following 7 days treatment with DMSO or Fenretinide, using TriZol reagent (Life Technologies) and purified (RNeasy Mini Kit, Qiagen) according to the manufacturers instructions. cDNA was synthesised using Superscript II and E.Coli DNA polymerase according to the manufacturers instructions (Life Technologies), purified (GeneChip® Sample Cleanup Module, Qiagen) and used as a template for the synthesis of biotin-labelled cRNA (GeneChip IVT Labeling Kit (Affymetrix). The biotin-labelled cRNA was purified and fragmented using the GeneChip® Sample Cleanup Module. cRNAs were hybridised to the Human U133A 2.0 Affymetrix Microarray. Data analysis was performed in R using packages from Bioconductor53 (link). Data sets for each array were quality assessed using affyQCReport. GenChip Robust Multiarray averaging (GC-RMA) was used to correct background, normalise data and convert fluorescence into expression levels. Differentially expressed genes were identified using a two sample T-test followed by FDR analysis.
3
Transcriptome Analysis of Rat Gene Expression
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The quality of RNA was determined by using RNA 6000 Nano LabChip Kit and Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). Preparation of cRNA was performed according to the protocol provided by Affymetrix (Santa Clara, CA). The total RNA from individual animal was further purified by using RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). Total RNA (3 μg) was converted to double-stranded cDNA using SuperScript System (Invitrogen, Carlsbad, CA) and an oligo (dT24) primer containing a T7 RNA polymerase promoter site. Biotin-labeled cRNA was synthesized from cDNA using a labeling kit and purified by using a GeneChip Cleanup Sample Module (Qiagen Inc., Valencia, CA, USA). The yield of the in vitro transcription reaction was determined by product absorbance at 260 nm measured by NanoDrop ND-1000 (NanoDrop Technologies, Inc., Montchanin, DE), and a size of cRNA probes was evaluated by using RNA 6000 Nano LabChip Kit (Agilent, Palo Alto, CA, USA). Fragmented cRNA was used for hybridization to GeneChip® Rat Gene 2.0 ST arrays (Affymetrix). Arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidic Station 400 (Affymetrix), according to the standard protocol of the manufacturer. The arrays were scanned by using GeneChip Scanner 3000 (Affymetrix).
4
Agilent Bioanalyzer RNA Profiling
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The quality of RNA was determined by using an RNA 6000 Nano LabChip Kit and Agilent Bioanalyser 2100 (Agilent, Palo Alto, CA, USA). Preparation of cRNA was performed according to the protocol provided by Affymetrix (Santa Clara, CA). The same amounts of total RNA from two animals were pooled and further purified by using an RNeasy Mini Kit (Qiagen Inc. Total RNA (3 μg) derived from each pool (n = 4) was converted to double-stranded cDNA using a SuperScript System (Invitrogen, Carlsbad, CA) and an oligo(dT)24 primer containing a T7 RNA polymerase promoter site. Biotin-labelled cRNA was synthesised from cDNA using a labelling Kit and purified by using a GeneChip Cleanup Sample Module (Qiagen Inc., Valencia, CA, USA). The yield of the in vitro transcription reaction was determined by the product absorbance at 260 nm, as measured by NanoDrop ND-1000 (NanoDrop Technologies, Inc., Montchanin, DE), size of cRNA probes was evaluated by using RNA 6000 Nano LabChip Kit (Agilent, Palo Alto, CA, USA). Fragmented cRNA was used for hybridization to GeneChip® Rat Gene 2.0 ST arrays (Affymetrix). The arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidics Station 400 (Affymetrix) according to the standard protocol of the manufacturer. The arrays were scanned by using a GeneChip Scanner 3000 (Affymetrix).
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