For ten of the 12 species (all except A. amphitrite and T. j. formosana, for which transcriptome data had already been published), tissue samples (single individual of each species) of the prosoma and base/peduncle (where the cement gland is located) were carefully isolated without contaminating each other. The total RNA of the prosoma and base was extracted using TRIzol® Reagent (Invitrogen, Camarillo, CA) and High Pure RNA Isolation Kit (Roche Applied Science, Germany), respectively. RNA quality assessment was conducted by a Bioanalyzer 2100 with RNA 6000 labchip kit (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries for both parts of all 10 species were prepared using Illumina TruSeq RNA Sample Prep Kits v2 and subsequently sequenced by HiSeq™ 2500 High-Throughput Mode v4 with paired-end 125 base-pair reads located at the High throughput Genomics Core, Biodiversity Research Center, Academia Sinica, Taiwan according to the manufacturer’s instructions (Illumina, San Diego, CA).
Rna 6000 labchip kit
Manufactured by Agilent Technologies
Sourced in United States, Canada
The RNA 6000 LabChip kit is a laboratory tool used for the analysis of RNA samples. It provides a automated and sensitive method for the assessment of RNA integrity and concentration.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (33 mentions)
2100 bioanalyzer, by Agilent Technologies (30 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (27 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (5 mentions)
Ampure xp bead, by Beckman Coulter (5 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Sureselect strand specific rna library preparation kit, by Beckman Coulter (4 mentions)
Truseq rna sample prep kits v2, by Illumina (3 mentions)
Nucleospin rna clean up kit, by Macherey-Nagel (3 mentions)
Microdismembrator, by B. Braun (3 mentions)
Rnase free dnase 1, by Qiagen (3 mentions)
Tri reagent, by Thermo Fisher Scientific (3 mentions)
Agilent 2100, by Agilent Technologies (3 mentions)
Agilent 2100 expert software, by Agilent Technologies (3 mentions)
Hiseq 2500 high throughput mode v4, by Illumina (2 mentions)
Trizol plus rna purification kit, by Merck Group (2 mentions)
Libra s22, by Harvard Bioscience (2 mentions)
Targetamp nano labeling kit for expression beadchip, by Illumina (2 mentions)
74 protocols using rna 6000 labchip kit
1
Transcriptome Analysis of Barnacle Species
2
Robust RNA Isolation from Tissues
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For RNA isolation, tissue samples were transferred into lysing matrix tubes (MP Biomedicals, Illkirch, France) containing an appropriate buffer and were homogenized in a FastPrep® instrument (MP Biomedicals) for 30 s. Lysates were centrifuged for 3 min at 12,000 g to eliminate excess tissue debris. The RNA was isolated by the RNeasy Mini Kit (Qiagen, Madrid, Spain) following the manufacturer’s recommendations and then quantified with a NanoDrop spectrophotometer and NanoDrop IVD-1000 v.3.1.2 software (NanoDrop Technologies, Wilmington, DE, USA). The Agilent 2100 Bioanalyzer with the RNA 6000 LabChip kit (Agilent Technologies, Madrid, Spain) was used to provide an RNA integrity number for each sample. All samples used for further experiments showed an RNA integrity number (RIN) ≥ 9 and purity between 1.885 and 2.042 using the A260/A280 ratio.
3
RNA Isolation and Microarray Analysis of Aspergillus niger
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RNA isolation and microarray hybridization were performed as described previously [59 (link)]. In brief, RNA for microarray analysis was extracted using TRIzol reagent (Invitrogen) and purified using TRIzol® Plus RNA Purification Kit (Sigma-Aldrich) according to the instructions of the manufacturer. The concentration of RNA was calculated from the absorbance at 260 nm in a spectrophotometer (Biochrom Libra S22). The quality of the RNA was analyzed with an Agilent 2100 Bioanalyzer using a RNA6000 LabChip kit (Agilent Technology). Microarray hybridization using the Affymetrix GeneChips A. niger Genome Array was performed at GenomeScan (Leiden, The Netherlands).
4
Transcriptomic Analysis of HBCD Exposure
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Total RNA was extracted from powdered brain tissue of HBCD exposed and control mice (n = 6 per group) as described by us before (Rasinger et al. 2014 (link)). In short, total RNA was extracted using Trizol (Invitrogen, Life Technologies, USA) and the Qiagen RNeasy-Mini kit (Qiagen, Canada). RNA purity was assessed using a Nanodrop ND-100 UV–Vis Spectrophotometer (Nanodrop Technologies, USA) and RNA quality and integrity were assessed using the Agilent 2100 Bioanalyzer in combination with the RNA 6000 LabChip kit (Agilent Technologies, USA). Gene expression microarray analysis was performed using GeneChip Mouse Exon 1.0 ST arrays (Affymetrix, USA) following the manufacturer’s instructions. Array images were acquired using a GeneChip Scanner 3000 (Affymetrix, USA). Array data were normalised using RMAsketch as implemented in the Affymetrix Expression Console.
5
Gene Expression Changes in Mouse Otitis Media
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Changes in gene expression in the ME during the course of OM in mice was
evaluated using DNA microarrays, as described elsewhere.34 (link) Briefly, WT C57Bl/6:CB F1 hybrid mice (60–90 d old) were purchased from
Jackson Laboratories (Bar Harbor, ME). Twenty mice per time point were
inoculated bilaterally with NTHi strain 3655. The ME mucosae
were harvested at different intervals: 0 (no treatment control), 3 h, 6 h, and
1, 2, 3, 5, and 7 d after NTHi infection. Sham-inoculated (saline) animals
served as additional controls. Total RNA was extracted using TRIzol (Invitrogen,
CA). The RNA quality was assessed using the RNA 6000 Labchip Kit on the Agilent
2100 Bioanalyzer to ensure the integrity of 18S and 28S ribosomal RNA. Reverse
transcription of the mRNA was done using a T7-oligodT primer and T7 RNA
polymerase to generate biotinylated cRNA probes that were hybridized onto two
Affymetrix MU430 2.0 microarrays per time point sample. The procedures were then
duplicated for each time point to obtain a second, independent replication. The
raw data of gene expression levels were median normalized and statistical
differences in gene transcript expression levels were evaluated using a
variance-modeled posterior inference approach (VAMPIRE).35 (link) Individual transcript fold-level changes were visualized using Genespring
(Agilent Technologies, Santa Clara, CA).
evaluated using DNA microarrays, as described elsewhere.34 (link) Briefly, WT C57Bl/6:CB F1 hybrid mice (60–90 d old) were purchased from
Jackson Laboratories (Bar Harbor, ME). Twenty mice per time point were
inoculated bilaterally with NTHi strain 3655. The ME mucosae
were harvested at different intervals: 0 (no treatment control), 3 h, 6 h, and
1, 2, 3, 5, and 7 d after NTHi infection. Sham-inoculated (saline) animals
served as additional controls. Total RNA was extracted using TRIzol (Invitrogen,
CA). The RNA quality was assessed using the RNA 6000 Labchip Kit on the Agilent
2100 Bioanalyzer to ensure the integrity of 18S and 28S ribosomal RNA. Reverse
transcription of the mRNA was done using a T7-oligodT primer and T7 RNA
polymerase to generate biotinylated cRNA probes that were hybridized onto two
Affymetrix MU430 2.0 microarrays per time point sample. The procedures were then
duplicated for each time point to obtain a second, independent replication. The
raw data of gene expression levels were median normalized and statistical
differences in gene transcript expression levels were evaluated using a
variance-modeled posterior inference approach (VAMPIRE).35 (link) Individual transcript fold-level changes were visualized using Genespring
(Agilent Technologies, Santa Clara, CA).
6
Duodenal Biopsy RNA Extraction and Microarray
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For study purpose, one biopsy (15–20 mg) was taken from the descending duodenum of each patient. Fresh tissue samples were snap frozen and stored in liquid nitrogen until preparation. Frozen biopsies were disrupted and homogenized by TissueLyzer from Quiagen (Hilden, Germany). Total RNA was isolated using AllPrep® DNA/RNA Micro kit (QIAGEN, Hilden, Germany) and stored at −70 °C.
Before microarray analysis RNA integrity and concentration was examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6.000 LabChip Kit (Agilent Technologies) according to manufacturer’s instructions. Then, 250 ng RNA per sample was ethanol precipitated with GlycoBlue (Invitrogen) as carrier and dissolved at a concentration of 100–150 ng/µL prior to probe synthesis using the TargetAmp™- Nano Labeling Kit for Illumina Expression BeadChip (Epicentre Biotechnologies, Madison, WI, USA). From each probe, 750 ng of cRNA were hybridized to Human HT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) and scanned on the Illumina HiScan instrument according to the manufacturer’s specifications.
Before microarray analysis RNA integrity and concentration was examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6.000 LabChip Kit (Agilent Technologies) according to manufacturer’s instructions. Then, 250 ng RNA per sample was ethanol precipitated with GlycoBlue (Invitrogen) as carrier and dissolved at a concentration of 100–150 ng/µL prior to probe synthesis using the TargetAmp™- Nano Labeling Kit for Illumina Expression BeadChip (Epicentre Biotechnologies, Madison, WI, USA). From each probe, 750 ng of cRNA were hybridized to Human HT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) and scanned on the Illumina HiScan instrument according to the manufacturer’s specifications.
7
RNA Extraction from Mycelium
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Mycelium was ground using a microdismembrator (B. Braun GmBh, Melsungen, Germany), and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. The RNA was purified using a Nucleospin RNA cleanup kit (Macherey-Nagel GmBh, Düren, Germany). The concentration of RNA was measured at A260. The quality of the RNA was analyzed with an Agilent 2100 bioanalyzer, using an RNA6000 LabChip kit (Agilent Technology, Palo Alto, CA).
8
Genomic Profiling of Glioblastoma Multiforme
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Sixty GBM samples with OS and PFS information were obtained during surgical resection from Tian Tan Hospital from 2008 to 2010. All donors signed informed consent forms. The use of human tissue samples and the experimental procedures for this study were reviewed and approved by the Ethics Committee of the Cancer Institute and Hospital, Chinese Academy of Medical Sciences.
Total RNA was isolated with Trizol reagent (Invitrogen, CA, USA), and those allocated for microarray detection were purified with an RNeasy kit (Qiagen, MD, USA). RNA was quantitated with ND-1000 UV-VIS Spectrophotometer (NanoDrop Technologies, DE, USA) and the integrity of RNA was assessed using the RNA 6000 Labchip kit in combination with the Agilent 2100 Bioanalyzer (Agilent, CA, USA). Purified total RNA samples were labeled and hybridized to Agilent 4 × 44 K Whole Human Genome Oligo Microarrays according to the manufacturer's instructions.
Furthermore, the raw and processed gene expression data and clinical information of the sixty GBM samples have been deposited in Gene Expression Omnibus (GEO) database with the series accession numbers GSE74187.
Total RNA was isolated with Trizol reagent (Invitrogen, CA, USA), and those allocated for microarray detection were purified with an RNeasy kit (Qiagen, MD, USA). RNA was quantitated with ND-1000 UV-VIS Spectrophotometer (NanoDrop Technologies, DE, USA) and the integrity of RNA was assessed using the RNA 6000 Labchip kit in combination with the Agilent 2100 Bioanalyzer (Agilent, CA, USA). Purified total RNA samples were labeled and hybridized to Agilent 4 × 44 K Whole Human Genome Oligo Microarrays according to the manufacturer's instructions.
Furthermore, the raw and processed gene expression data and clinical information of the sixty GBM samples have been deposited in Gene Expression Omnibus (GEO) database with the series accession numbers GSE74187.
9
RNA Isolation and Real-Time PCR Analysis
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Total RNAs were extracted with Qiagen RNeasy Isolation Kit (Qiagen, Venlo, Netherlands), treated with RNase-free DNase I (Qiagen), and quantified by NanoDrop (ND-1000) spectrophotometer (NanoDrop, Wilmington, DE, USA). The RNA quality was evaluated on an Agilent 2100 bioanalyzer using the RNA 6000 Labchip kit (Agilent Technologies, Palo Alto, CA, USA). cDNA synthesis was performed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA), as recommended by the manufacturer. Real-time PCR was performed by predesigned primer/probe TaqMan gene expression assays (Applied Biosystems) and normalized to HPRT housekeeping gene (Additional file 2 ). Experiments were assessed in triplicate at least using TaqMan Fast-Univ Master Mix (Applied Biosystems) using an Applied Biosystems StepOnePlus Real-Time PCR System and analysed by StepOne Software v2.2.2 with a two-step PCR protocol as previously described [9 (link)]. The comparative ΔCt method was used for relative quantification of gene expression on duplicate of each reaction.
10
Brain Region Transcriptome Analysis
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The total RNA of homogenized tissue from specific brain regions was isolated using the Trizol® method according to manufacturer’s instructions (GibcoBRL) and its concentration was determined using the nanodrop ND-1000 spectrophotometer followed by its quality and integrity measurement by electrophoresis on RNA 6000 LabChip® kit (Agilent 2100 Bioanalyzer). The RNA was transcribed into the cDNA using the iScript cDNA Synthesis Kit (BioRad). The primer pairs used for RT-PCR are the same used previously for quantitative RT-PCR (Vogelgesang et al., 2017 (link)).
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