RNA was extracted using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. Quality of RNA was analyzed using the Bioanalyzer platform (Agilent Technologies). Two micrograms of total RNA were treated according to standard Affymetrix eukaryotic RNA labeling protocols (Affymetrix, Santa Clara, CA). Fifteen micrograms of biotin labeled cRNA was fragmented according to Affymetrix eukaryotic sample protocol. Hybridization, staining, and washing of the Affymetrix HG-U133_Plus_2 Arrays were performed using the Affymetrix Fluidics Station 450 and Hybridization Oven 640 under standard conditions. The raw data were processed using the GCRMA-normalization method [18 (link)].
Bioanalyzer platform
Manufactured by Agilent Technologies
Sourced in United States
The Bioanalyzer platform is a lab equipment product from Agilent Technologies. It is designed to perform automated, high-resolution analysis of DNA, RNA, and proteins. The platform uses microfluidic technology to enable rapid, sensitive, and quantitative assessments of sample integrity and concentration.
Automatically generated - may contain errors
Lab products found in correlation
Hybridization oven 640, by Thermo Fisher Scientific (1 mentions)
Fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Truseq rna, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Rna 6000 nano chip, by Agilent Technologies (1 mentions)
Linear acrylamide, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit column, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription system, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Promega (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Qubit dsdna hs assay kit, by Illumina (1 mentions)
Nextseq platform, by Illumina (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Msb spin pcrapace purification kit, by Stratec (1 mentions)
Miseq qc run, by Illumina (1 mentions)
Truseq dna library preparation kit, by Illumina (1 mentions)
7 protocols using bioanalyzer platform
1
Affymetrix Gene Expression Analysis
2
RNA Extraction and Sequencing
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Total RNA was isolated with the RNeasy Kit (Qiagen) following the manufacturer’s instructions. The integrity of RNA was assessed with RNA 6000 Nano chip with a Bioanalyzer platform (Agilent), and only samples with RNA integrity number (RIN) ≥8 were included. RNA samples were used for constructing cDNA libraries with TruSeq RNA (Illumina) following the manufacturer’s protocol. Libraries were sequenced in an Illumina HiSeq 2500 platform to produce 2×100 bp reads.
3
Bovine Muscle Tissue RNA Extraction
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These procedures were exactly as described previously.13 Briefly, muscle tissue sample was weighted (~0.3–0.5 g) and immediately homogenized with Trizol reagent (Invitrogen Corp.) and linear acrylamide (Ambion® Cat. No. 9520) as coprecipitant to proceed with RNA extraction. Genomic DNA was removed from RNA with DNase using RNeasy Mini Kit columns (Qiagen, Germany). The RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The purity of RNA (A260/A280) for all samples was above 1.81. Moreover, RNA integrity number (RIN) was measured using the Bioanalyzer platform (Agilent Technologies, Inc., Santa Clara, CA, USA). The final data were normalized using the geometric mean of UXT, MTG1 and RPS15A, which were validated as suitable internal control genes in bovine LM. During the analysis of the qPCR results, it was determined that WNT10b and MLXIPL had a very low level of expression; thus, we concentrated the cDNA (dilution 1:3) and reanalyzed those genes along with the internal controls.
4
Quantitative Analysis of Exon Skipping
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Isolated RNAs were treated with DNase I (Promega or Zym Research) to degrade contaminating DNA following the company's recommendations. 0.5–1.0 µg of DNase-treated total RNA was used as template for reverse transcription using the High Capacity cDNA Reverse Transcription System (Applied Biosystems) and random hexamer primers. PCR reactions were performed using the Taq polymerase with PCR primers for the minigene constructs complementary to T7 (forward) and SP6 (reverse) promoter sequences present in the construct, respectively (Gregory et al. 2007 (link)). PCR primers for endogenous IL7R were complementary to exon 5 (forward) and exon 7 (reverse). PCR Primers for endogenous IL7R in human and nonhuman primate samples, were complementary to exon 5 (forward) and exon 6 (reverse) sequences identical between all species. PCR primers for the GFP-IL7R reporter were complementary to GFP sequences (forward and reverse) (described in Somarelli et al. 2013 (link)). PCR products were quantified using the Bioanalyzer platform (Agilent). Percentage exon 6 skipping was determined as: [(skipped product)/(included + skipped products) × 100]. The data are presented as the mean of triplicate samples and error bars represent standard deviations. Statistical significance in knockdown and reporter experiments was assessed using Student's t-test (two-sided).
5
UPEC Infection RNA Isolation
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Mice infected with 108 cfu of UPEC or PBS were humanely euthanized at indicated time points. All mock-infected mice were euthanized at 24 hpi. Bladders were aseptically harvested, flash-frozen in liquid nitrogen, and stored at −80°C. RNA isolation from whole bladders was performed using the with QIAGEN RNeasy Plus kit (74136). RNA quality was spot-checked using the Bioanalyzer platform (Agilent) and all RNA integrity number (RIN) scores were 8.9 or higher.
6
Whole Transcriptome Amplification and RNA-Seq
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cDNA synthesis and PCR amplification was performed according to the modified version of the Whole Transcriptome Amplification (WTA2, SigmaAldrich, St. Louis, MO, USA) kit [45 (link)]. Amplicons were purified using the MSB® SPIN PCRAPACE purification kit (Stratec Biomedical, Birkenfeld, Germany), according to manufacturer’s protocol. The concentration of the purified DNA was quantified using the Qubit dsDNA HS assay kit, according to the manufacturer’s protocol and then diluted to 1.2 ng/µL.
DNA libraries were prepared using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) as per the manufacturer’s instructions, and libraries were purified following the Agencourt® AMPure® PCR purification protocol. The concentration of the purified library was quantified using a Qubit dsDNA HS assay kit, according to the manufacturer’s instructions. Then, the DNA library size distribution and quality were evaluated using an hsDNA chip on a Bioanalyzer platform (Agilent Technologies, 2009, Santa Clare, CA, USA). Sequencing was performed using the Illumina NextSeq platform for paired-end sequencing with a base length of 2 × 150 bp, and an average of 9,304,652 reads were retrieved per sample.
DNA libraries were prepared using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) as per the manufacturer’s instructions, and libraries were purified following the Agencourt® AMPure® PCR purification protocol. The concentration of the purified library was quantified using a Qubit dsDNA HS assay kit, according to the manufacturer’s instructions. Then, the DNA library size distribution and quality were evaluated using an hsDNA chip on a Bioanalyzer platform (Agilent Technologies, 2009, Santa Clare, CA, USA). Sequencing was performed using the Illumina NextSeq platform for paired-end sequencing with a base length of 2 × 150 bp, and an average of 9,304,652 reads were retrieved per sample.
7
Arabidopsis DNA Isolation and Sequencing
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For Col-0, polIb, reca1, why1why3, reca1polIb, why1why3polIb, and why1why3reca1, total DNA was isolated from ≈400 mg pools of 14-d-old Arabidopsis plants using the cetyl trimethyl-ammonium DNA extraction protocol (Weigel and Glazebrook 2002 ). DNA was fragmented to ≈200–500 bp using S-Series Covaris according to Illumina's specifications. Libraries were prepared using the TruSeq DNA library preparation kit (Illumina) according to the manufacturer's instructions. Efficient library generation was then assessed using a Bioanalyzer platform (Agilent), and an Illumina MiSeq-QC run was performed. Sequencing was performed using an Illumina HiSeq 2000 using TruSeq SBS v3 chemistry at the Institute for Research in Immunology and Cancer's Genomics Platform (Université de Montréal). Cluster density was targeted at around 600–800 kilo-clusters mm–2.
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