This experimental system was totally based on Affymetrix DNA Microarray Guide. Total RNA of FOXP3 over-expressed HepG2 was isolated with TRIzol (Invitrogen, USA) according to the manufacturer’s protocols and purified by RNeasy mini Kit (Qiagen, Germany), then Affymetrix one-cycle cDNA Synthesis Kit (Affymetrix, USA) was used to synthesis cDNA, then purified by Affymetrix GeneChip Sample Cleanup Module (Affymetrix, USA). Hybridization, elution and staining of chip were followed untill synthesis (GeneChip IVT Labeling Kit, Affymetrix, USA), purification (GeneChip Sample Cleanup Module, Affymetrix, USA) and fragmentation of cRNA were done. And the data was analyzed after scanning chip.
Genechip sample cleanup module
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip Sample Cleanup Module is a laboratory equipment designed to purify and concentrate nucleic acid samples prior to analysis. It utilizes a solid-phase extraction method to remove contaminants and unwanted components from the sample, preparing it for downstream applications such as gene expression analysis or sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Genechip scanner 3000, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Human genome u133 plus 2.0 array, by Thermo Fisher Scientific (3 mentions)
Genechip ivt labeling kit, by Thermo Fisher Scientific (3 mentions)
Bioarray highyield rna transcript labeling kit, by Enzo Life Sciences (3 mentions)
Gcos 1, by Thermo Fisher Scientific (3 mentions)
Genechip operating software, by Thermo Fisher Scientific (3 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Affymetrix cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Bioarray high yield rna transcript labeling system, by Enzo Life Sciences (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
One cycle cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Fluidics station 400, by Thermo Fisher Scientific (2 mentions)
Agilent gene array scanner g2500a, by Agilent Technologies (2 mentions)
Hg u133 plus 2.0 genechip oligonucleotide arrays, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Human gene chip exon1.0 st array, by Thermo Fisher Scientific (1 mentions)
Ribominus human mouse transcriptome isolation kit, by Thermo Fisher Scientific (1 mentions)
Genechip human exon 1.0 st array, by Thermo Fisher Scientific (1 mentions)
19 protocols using genechip sample cleanup module
1
FOXP3 Overexpression in HepG2 Transcriptome
2
Genome-wide Expression Analysis of Arabidopsis Mutants
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For genome-wide expression analysis of mutants, OX lines, KO lines, and the wild-type, total RNA was extracted from calli using an RNeasy Plant Mini Kit (Qiagen), and then quantified using Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). Two micrograms of total RNA was subjected to cDNA synthesis by One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA, USA), followed by purification by GeneChip Sample Cleanup Module (Affymetrix) and labeling with biotin by GeneChip IVT Labeling Kit (Affymetrix). The resultant labeled cRNA was further purified by GeneChip Sample Cleanup Module (Affymetrix), and hybridized with GeneChip Arabidpsis ATH1 Genome Array (Affymetrix) for 16 h according to the manufacturer’s protocol. The hybridized chips were washed and stained using Fluidics Station 450 (Affymetrix) by 49-Format program, and read by Affymetrix GeneChip Scanner 3000 (Affymetrix). Results were analyzed using GeneChip Operating Software (Affymetrix) and with the Partek Genomics Suite 6.6 (St. Louis, MO, USA).
3
Comprehensive Transcriptomic Analysis Using Affymetrix Exon Arrays
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Total RNA was reverse-transcribed and hybridized onto Affymetrix HG-U133 Plus 2.0
array, using the identical protocol and ID names for genes as GeneChip Human Exon
1.0ST Arrays (Affymetrix, Santa Clara, CA, USA). The Affymetrix Human Gene Chip Exon
1.0 ST Array interrogates over one million exons representing over 17,868 NCBI
Reference Sequence (RefSeq) transcripts. Arrays were run using the
manufacturer's technical protocol (Affymetrix, Santa Clara, CA). Briefly, 2 qg
of total RNA was subjected to a ribosomal RNA removal procedure (RiboMinus Human/
Mouse Transcriptome Isolation Kit, Invitrogen - Thermo Fischer Scientific) to reduce
the 28S and 18S rRNA population to minimize background and increase sensitivity of
the assay. Reduced RNA was reverse-transcribed to cDNA using random hexamers tagged
with a T7 promoter sequence followed by a second strand cDNA synthesis using DNA
polymerase (GeneChip WT cDNA Synthesis Kit, Affymetrix). The resulting
double-stranded cDNA was used for amplification of antisense cRNA and cleaned using
the Gene Chip Sample Cleanup Module (Affymetrix). A second cycle cDNA synthesis was
performed using random primers to reverse transcribe the cRNA into sense single
stranded DNA, which was fragmented, labeled, and hybridized to arrays. Arrays were
washed, stained, and scanned on the Affymetrix Fluidics Station and G7 Affymetrix
high-resolution scanner using GCOS 1.3.
array, using the identical protocol and ID names for genes as GeneChip Human Exon
1.0ST Arrays (Affymetrix, Santa Clara, CA, USA). The Affymetrix Human Gene Chip Exon
1.0 ST Array interrogates over one million exons representing over 17,868 NCBI
Reference Sequence (RefSeq) transcripts. Arrays were run using the
manufacturer's technical protocol (Affymetrix, Santa Clara, CA). Briefly, 2 qg
of total RNA was subjected to a ribosomal RNA removal procedure (RiboMinus Human/
Mouse Transcriptome Isolation Kit, Invitrogen - Thermo Fischer Scientific) to reduce
the 28S and 18S rRNA population to minimize background and increase sensitivity of
the assay. Reduced RNA was reverse-transcribed to cDNA using random hexamers tagged
with a T7 promoter sequence followed by a second strand cDNA synthesis using DNA
polymerase (GeneChip WT cDNA Synthesis Kit, Affymetrix). The resulting
double-stranded cDNA was used for amplification of antisense cRNA and cleaned using
the Gene Chip Sample Cleanup Module (Affymetrix). A second cycle cDNA synthesis was
performed using random primers to reverse transcribe the cRNA into sense single
stranded DNA, which was fragmented, labeled, and hybridized to arrays. Arrays were
washed, stained, and scanned on the Affymetrix Fluidics Station and G7 Affymetrix
high-resolution scanner using GCOS 1.3.
4
Microarray-based Gene Expression Analysis
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Hybridization targets for the GeneChip Human Gene 1.0 ST array were prepared using the GeneChip WT cDNA Synthesis and Amplification Kit, GeneChip Sample Cleanup Module and GeneChip WT Terminal Labeling Kit according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, the cells were harvested in the logarithmic growth phase and total RNA was extracted. Total RNA (100 ng) was converted into double-stranded cDNA (1st-cycle), and the complementary RNA (cRNA) was synthesized by in vitro transcription. After purification and measurement of cRNA, 10 μg was converted into single-stranded DNA (ssDNA, 2nd cycle), of which 5.5 μg was fragmented and labeled. The ssDNA was hybridized to the array described above for 16 hours at 45°C. Following hybridization, the array was automatically washed and stained with the GeneChip Hybridization, Wash and Stain Kit. The Probe Array was scanned using the GeneChip Scanner 3000 7G. Microarray analysis was performed three times using three independent cell cultures.
5
Microarray Analysis of ESC-Derived Hematopoietic Progenitors
6
Genome-wide gene expression analysis
7
Total RNA Extraction and Microarray Preparation
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Total RNA was isolated from each sample using Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA). All RNA samples exhibited intact 28S and 18S ribosomal RNA on denaturing agarose gel electrophoresis and OD260/OD280 absorbance ratio fell within the acceptable range of 1.8–2.1. RNA yield was determined spectrophotometrically (Nanodrop ND-1000 spectrophotometer; Thermo scientific, Waltham, MA, USA). RNA extracts were DNase treated. For array analysis at least 3 ug of total RNA was transcribed into Double Stranded cDNA with oligo (dT)24 T7 primer using Invitrogen SuperScript Double-Stranded cDNA synthesis kit (Life Technologies, Grand Island, NY, USA). The cDNA was purified with Affymetrix GeneChip Sample Cleanup module (Affymetrix, Santa Clara, CA, USA) and used into an in vitro transcription reaction to produce labeled cRNA with Enzo Bioarray High Yield RNA Transcript Labeling Kit from Enzo Life Sciences (Farmingdale, NY, USA) and further fragmented to 35–200 bp oligos for hybridization. All steps for sample preparation and processing were performed according to manufacturer's instructions.
8
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.
9
Transcriptional Response of Salt-Tolerant Wheat to NaCl Treatment
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Plants of the salt-tolerant wheat line RH8706-49 were treated with 135 mM NaCl for 0, 1, 6, 12, 72 h, respectively. The roots were then taken for total RNA preparation using the TRIzol (Invitrogen) reagent. Total RNA was purified with the RNeasy Mini kit (Qiagen). Double-stranded cDNA was synthesized with the one-cycle cDNA Synthesis Kit (Affymetrix), and then purified with the GeneChip Sample Cleanup Module (Affymetrix). The purified cDNA was used to prepare biotin-labeled cRNA using a GeneChip IVT Labeling Kit, according to the manufacturer’s instructions. The biotin-labeled cRNA was fragmented at 94°C for 35 min, which yielded the targets used for hybridization. The targets were hybridized with the Affymetrix Wheat Genome Array P/N:520254, and washing and scanning were carried out according to the assay procedure. The hybridization image was analyzed with Affymetrix Microarray Suite 5.0 software and the data were normalized. Clustering analysis was carried out with the Cluster and Tree View software.
10
Transcriptome Analysis of MDA-MB-231 Cells under Hypoxic Conditions
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