Total RNA was isolated by using RNeasy MICROKit (Qiagen GmbH, Hilden, Germany) following manufacturer’s recommendations. Total RNA concentration and quality control was assessed using RNA 6000 pico kit (Agilent, Santa Clara, CA, USA). Due to the small amount of RNA obtained, RNA samples were amplified by using Ovation PicoSL System V2 (NuGene Technologies, CA, USA) and ENCORe Biotin module (NuGene technologies). RNA MinElute Reaction Cleanup Kit (Qiagen) was used to purify amplified RNA. RNA samples were labeled and hybridized by using GeneChip Hybridization Control Kit and GeneChip Hybridization, Wash and stain Kit HT (Affymetrix, Santa Clara, CA, USA), respectively. Labeled RNA samples were then hybridized to Affymetrix HG-U219 genechips (16 arrays plate) (Affymetrix) according to the manufacturer’s instructions. Data normalization and analysis was performed using GeneSpring GX12 (Agilent, Santa Clara, CA) as described previously [71 (link)]. For detection of differentially expressed genes, a P-value cut-off of 0.05 was used in combination with a fold-change cut-off of 2.0. Raw data are made publically available on the NCBI Gene Expression Omnibus database, with accession number GSE68000.
Genechip hybridization control kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip Hybridization Control Kit is a set of RNA controls used in gene expression analysis experiments on Affymetrix GeneChip arrays. The kit contains a mixture of in vitro transcribed, polyadenylated RNAs that can be spiked into sample RNA prior to array hybridization. These controls are used to monitor the hybridization, washing, staining, and scanning steps of the GeneChip experiment.
Automatically generated - may contain errors
Lab products found in correlation
Flashtag biotin hsr rna labeling kit, by Thermo Fisher Scientific (3 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (2 mentions)
Genechip mirna 4.0 array, by Thermo Fisher Scientific (2 mentions)
Microarray system, by Thermo Fisher Scientific (2 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (2 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Rna 6000 pico kit, by Agilent Technologies (1 mentions)
Genespring gx 12, by Agilent Technologies (1 mentions)
Fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Genechip command console software, by Thermo Fisher Scientific (1 mentions)
Genechip mirna 4, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Genechip operating software, by Thermo Fisher Scientific (1 mentions)
Bioarray highyield rna transcript labeling kit t7, by Enzo Life Sciences (1 mentions)
5 protocols using genechip hybridization control kit
1
Whole-Transcriptome Profiling by Microarray
2
Ovine Plasma miRNA Profiling
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The profile of miRNAs from ovine plasma samples was investigated using the Affymetrix Microarray system with the GeneChip miRNA 4.0 Array (Affymetrix, United States) that is arranged to retrieve mature miRNA sequences in miRBase (20.0) (http://mirbase.org/ftp.shtml ). Mature miRNA sequences of one hundred fifty from sheep are demonstrated in miRBase (20.0). A total of one 1) microgram of RNA was labeled with a FlashTagTM Biotin HSR RNA Labeling Kit (Affymetrix, United States). Following RNA labeling, through a GeneChip Hybridization Control Kit (Affymetrix, United States), microarray chips were hybridized with agitation at 60 rpm for 15 h. Then, the chip arrays were washed and subsequently stained through a Fluidics Station 450 (Affymetrix, Santa Clara, California, United States) with AGCC Fluidics Control Software. Fluorescence was detected from the array chip with an Affymetrix® GeneChip Scanner 3000.
3
MicroRNA Profiling by Affymetrix Array
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The miRNA profile was determined and analyzed by GeneChip miRNA 4.0 Array (Affymetrix, USA) from the Affymetrix Microarray system (Affymetrix, USA).
Total RNA (1000 ng) was tailed by Poly(A) RNA polymerase, according to the manufacture's protocol of the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, USA). RNA ligase was used to connect biotin‐labeled signal molecules with the RNA, and the ligands and chips were hybridized using a GeneChip Hybridization Control Kit (Affymetrix, USA) at 60 rpm for 16 hours. After the chip hybridization, Fluidics Station 450 with AGCC Fluidics Control Software was used for washing and dyeing. Affymetrix Gene Chip Scanner 3000 scan chip results. The probe cell intensity files generated by the Affymetrix Gene Chip Command Console software were imported into probe‐level summarization files for data extraction. The chip scanned images and raw data are generated in Expression Console Software and Transcriptome Analysis Console software by the AGCC operating system. Algorithm Options: One‐Way Between‐Subject ANOVA (unpaired). Default Filter Criteria: (a) Fold Change (linear) <−2 or Fold Change (linear) >2; (b) ANOVA P‐value (Condition pair) <.05.
Total RNA (1000 ng) was tailed by Poly(A) RNA polymerase, according to the manufacture's protocol of the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, USA). RNA ligase was used to connect biotin‐labeled signal molecules with the RNA, and the ligands and chips were hybridized using a GeneChip Hybridization Control Kit (Affymetrix, USA) at 60 rpm for 16 hours. After the chip hybridization, Fluidics Station 450 with AGCC Fluidics Control Software was used for washing and dyeing. Affymetrix Gene Chip Scanner 3000 scan chip results. The probe cell intensity files generated by the Affymetrix Gene Chip Command Console software were imported into probe‐level summarization files for data extraction. The chip scanned images and raw data are generated in Expression Console Software and Transcriptome Analysis Console software by the AGCC operating system. Algorithm Options: One‐Way Between‐Subject ANOVA (unpaired). Default Filter Criteria: (a) Fold Change (linear) <−2 or Fold Change (linear) >2; (b) ANOVA P‐value (Condition pair) <.05.
4
miRNA Profiling using Affymetrix Microarray
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The profiles of miRNAs was identified with the Affymetrix Microarray System. GeneChip miRNA 4.0 (Affymetrix, USA) commercial kit used in this system contains 30,434 mature miRNAs from 203 organisms and 2.025 pre-miRNA from humans, 2578 human mature miRNAs Release 20.0 miRBase set [16 (link)].
The following operations were performed sequentially with reference to the FlashTag Biotin HSR RNA Labeling Kit procedure. Total RNA (200 ng) was labeled with FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, USA). Then, array chips were hybridized with a GeneChip Hybridization Control Kit (Affymetrix, USA). Hybridization conditions were at 60 rpm, 48 °C for 16 h. The chips arrays were washed and stained on a Fluidics Station 450 with AGCC Fluidics Control Software. Fluorescence was scanned with an Affymetrix GeneChip 3000-7G Scanner [16 (link)].
The following operations were performed sequentially with reference to the FlashTag Biotin HSR RNA Labeling Kit procedure. Total RNA (200 ng) was labeled with FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, USA). Then, array chips were hybridized with a GeneChip Hybridization Control Kit (Affymetrix, USA). Hybridization conditions were at 60 rpm, 48 °C for 16 h. The chips arrays were washed and stained on a Fluidics Station 450 with AGCC Fluidics Control Software. Fluorescence was scanned with an Affymetrix GeneChip 3000-7G Scanner [16 (link)].
5
RNA Extraction and Microarray Analysis of iPSCs
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RNA was extracted from undifferentiated iPSCs; purified KP cells at days 3, 4, and 5, and CMs derived from KP cells at day 5 under serum condition (Figure S1B) using the RNeasy Mini Kit (Qiagen). Total RNA was reverse transcribed into cDNA via SuperScript II Reverse Transcriptase (Life Technologies). Samples were labeled with Cy3 using the BioArray HighYield RNA Transcript Labeling Kit (T7) (Enzo Life Sciences). Labeled cDNA was hybridized with Human Genome U133 Plus 2.0 Array by the GeneChip Hybridization Control Kit and the GeneChip Fluidics Station 450 (all Affymetrix) at 45 C for 16 hr. Arrays were scanned with the GeneChip Scanner 3000 7G according to the manufacturer's protocol and analyzed using GeneChip operating software (both Affymetrix). The raw data were normalized using the MAS5 algorithm (median score of the gene expressions: 500).
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