2100 bioanalyzer

Manufactured by Agilent Technologies

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The 2100 Bioanalyzer is a lab equipment product from Agilent Technologies. It is a microfluidic platform designed for the analysis of DNA, RNA, and proteins. The 2100 Bioanalyzer utilizes a lab-on-a-chip technology to perform automated electrophoretic separations and detection.

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Agilent Bioanalyzer 2100 DNA 1000 kit (3 citations) Agilent 2100 Bioanalyzer Expert High Sensitivity DNA Assay (2 citations) Agilent 2100 Bioanalyzer RNA Nano Chip (43 citations) Agilent 2100 Bioanalyzer Nanochip system (2 citations) Agilent 2100 Bioanalyzer II (2 citations) Agilent 2100 Bioanalyzer DNA12000 Chip (3 citations) 2100 Bioanalyzer High Sensitivity DNA Analysis Kit (2 citations) Agilent 2100 Bioanalyzer RNA assay (12 citations) Agilent 2100 Bioanalyzer Expert Software (2 citations) 2100 Bioanalyzer RNA Nano chip device (7 citations)

8 194 protocols using 2100 bioanalyzer

Sequencing Workflow for Total RNA-Seq

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Total RNA samples were normalized to the amount of RNA isolated from the corresponding IP sample. rRNA was then depleted from the RNA samples using the Ribominus™ Eukaryote Kit for RNA-Seq (Life Technologies). Depleted samples were ethanol precipitated, washed twice with 70% ethanol and resuspended in 10 μl DEPC water. rRNA depletion was checked on a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using a RNA nanochip and the remaining RNA stored at -80°C.
Sequencing libraries were generated using the whole Transcriptome Library Preparation protocol provided with the SOLiD® Total RNA-Seq Kit (Life Technologies). Briefly, rRNA depleted samples were fragmented using RNase III, and subsequently cleaned up using the RiboMinus™ Concentration Modules (Life Technologies). Fragmentation was assessed on a 2100 Bioanalyzer (Agilent Technologies) using the RNA picochip. Fragmented RNAs were reverse transcribed and size selected on a denaturing polyacrylamide gel selecting for 150 to 250 nucleotide cDNA. cDNA was then amplified and barcoded with SOLiD™ RNA Barcoding Kit. Samples were then purified using PureLink™ PCR Micro Kit (Life Technologies) and assessed on a 2100 Bioanalyzer (Agilent Technologies) using the High Sensitivity DNA chip. Samples were deposited on slides, and sequenced using the SOLiD v4 sequencing system (Life Technologies).

Costello J., Castelli L.M., Rowe W., Kershaw C.J., Talavera D., Mohammad-Qureshi S.S., Sims P.F., Grant C.M., Pavitt G.D., Hubbard S.J, & Ashe M.P. (2015). Global mRNA selection mechanisms for translation initiation. Genome Biology, 16(1), 10.

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Robust Small RNA Extraction and Analysis

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Total RNA enriched for small RNA, including miRNA was isolated from SCAT samples using the Qiagen miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendation. The quality and quantity of small RNA were assessed using an Agilent Small RNA kit on a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Samples were then pooled (4 pools with 4 individuals per group and day). Pooling of samples was performed at the RNA level by mixing randomly selected RNA samples extracted from independent biological samples from the same group and equal amounts of RNA from each vole, before library preparation.
Libraries were prepared with NEXTFLEX Small RNA-seq v3 Kits with UDIs for Illumina (PerkinElmer) according to the manufacturer’s recommendations. The quality of the libraries was assessed using the Highly Sensitive DNA assay and a 2100 Bioanalyzer (Agilent). A total of 12 multiplexed libraries were parallel sequenced for single-end of 50 bp using the rapid-run mode of the Illumina HiSeq 2500 system by the sequencing facility of the Genome Biology Institute, FBN, Dummerstorf, Germany.

Sadri H., Ghaffari M.H., Trakooljul N., Ceciliani F, & Sauerwein H. (2022). MicroRNA profiling of subcutaneous adipose tissue in periparturient dairy cows at high or moderate body condition. Scientific Reports, 12, 14748.

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RNA-seq Protocol for Prenatal and Postnatal Brain

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RNA-seq was performed as a service by BGI America for P0 brains or as described previously (Takahashi et al. 2015 (link)) for P7 brains. Briefly, total RNA was extracted with the miRNeasy minikit (Qiagen, 217004) according to the manufacturer's instructions. RNA integrity number (RIN) of total RNA was quantified by an Agilent 2100 Bioanalyzer using an Agilent RNA 6000 nanokit (Agilent, 5067-1511). Total RNA with RIN values of ≥9.5 were used for RNA-seq library preparation. mRNA was purified from 2 µg of total RNA by NEXTflex poly(A) beads (Bioo Scientific, 512981), subjected to fragmentation and first and second strand syntheses, and cleaned up by EpiNext beads (EpiGentek, P1063). Second strand DNA was adenylated, ligated, and cleaned up twice by EpiNext beads. cDNA libraries were amplified by PCR and cleaned up twice by EpiNext beads. cDNA library quality was quantified by a 2100 Bioanalyzer using an Agilent high-sensitivity DNA kit (Agilent, 5067-4626, ). Libraries of P0 and P7 brains were sequenced separately as 50-bp single ends on a BGISEQ-500 or BGI in-house sequencer or as 75-bp single ends on Illumina NextSeq 500, respectively.

Usui N., Araujo D.J., Kulkarni A., Co M., Ellegood J., Harper M., Toriumi K., Lerch J.P, & Konopka G. (2017). Foxp1 regulation of neonatal vocalizations via cortical development. Genes & Development, 31(20), 2039-2055.

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Rhesus Macaque Transcriptome Analysis

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For oligonucleotide array analyses, total RNA was isolated using the RNeasy RNA isolation kit (Qiagen, CA). The quality and quantity of the RNA were determined using a Bioanalyzer 2100 (Agilent technologies). The cRNA was then amplified using the Low Input Quick Amp labeling kit, one color (Agilent technologies). The quantity and quality of cRNA were evaluated by capillary electrophoresis using an Agilent Technologies 2100 Bioanalyzer and NanoDrop ND-1000. Probe labeling and microarray hybridizations were performed as described in the Agilent 60-mer oligo microarray processing protocol (Agilent Technologies, CA). Six hundred ng of each labeled RNA sample was hybridized to Agilent 8X60K rhesus macaque Macaca Mulatta custom 8*60K array (AMADID 045743, Agilent technologies). The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for hybridization and array washing. Arrays were scanned with an Agilent microarray G2505C scanner and image analysis was performed using Agilent Feature Extraction 11.0.1 Software. Raw images were analyzed using Agilent Feature Extraction software (version 9.5.3.1) and the GE1_1100_Jul11 extraction protocol. All arrays were required to pass Agilent QC flags.

Echebli N., Tchitchek N., Dupuy S., Bruel T., Peireira Bittencourt Passaes C., Bosquet N., Le Grand R., Bourgeois C., Favier B., Cheynier R., Lambotte O, & Vaslin B. (2018). Stage-specific IFN-induced and IFN gene expression reveal convergence of type I and type II IFN and highlight their role in both acute and chronic stage of pathogenic SIV infection. PLoS ONE, 13(1), e0190334.

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Robust Nucleic Acid Extraction for Sequencing

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Snap frozen mouse tumor tissues were powdered in liquid nitrogen. Nucleic acid extraction was based on the methods and reagents described in the operation manuals of RNeasy mini kit (QIAGEN, catalog no. 74104) and DNeasy Blood & Tissue kit (QIAGEN, catalog no. 69504). The integrity of the cleanup RNA was determined by Agilent RNA 6000 Pico chip (Agilent, catalog no. 5067-1513) and 2100 Bioanalyzer (Agilent) and quantified using NanoDrop 2000/2000c Spectrophotometer (Thermo Fisher Scientific). Only high-quality RNA samples (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RNA integrity number (RIN) ≥ 7, >500 ng) were used to construct sequencing library. The integrity of the total DNA was determined by 2100 Bioanalyzer (Agilent) and quantified using NanoDrop 2000/2000c Spectrophotometer (Thermo Fisher Scientific). High-quality DNA samples (OD260/280 = 1.8–2.0, OD260/230 ≥ 2.0, DNA integrity number (DIN) ≥ 7, >500 ng) were used to construct sequencing library.

Qian W., Chen X., Sheng Y., Zhang L., Wang J., Song Z., Li Q.X, & Guo S. (2022). Tumor Purity in Preclinical Mouse Tumor Models. Cancer Research Communications, 2(5), 353-365.

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RNA Extraction from Woody Plants

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RNA extraction was performed according to Chang’s protocol [24 ] that is specific to woody plants. Briefly, 1 g of fresh tissue was grounded to a fine powder by using liquid nitrogen. Then, two extractions were performed with chloroform and RNA was precipitated with LiCl₂, extracted for a second time with chloroform and finally precipitated with ethanol. The obtained RNA was resuspended in 50 μl of DEPC (diethylpyrocarbonate) treated water and was quantified using a Nanodrop 1,000 spectrophotometer. The quality was assessed with a 2,100 Bioanalyzer (Agilent Technologies Inc.). Total RNA was purified using Poly (A) Purist kit (Ambion) and the quality was assessed again with a 2,100 Bioanalyzer (Agilent Technologies). cDNA was synthesized using cDNA Kit (Roche) for constructing a shotgun library. Roche 454 GS FLX Titanium sequencing platform at INDEAR (Rosario Agro Biotechnology Institute, http://webservices.indear.com/) in Rosario, Argentina performed sequencing. Moreover, all bioinformatic analysis was performed in the Bioinformatics Unit at the Biotechnology Institute at INTA.

Torales S.L., Rivarola M., Gonzalez S., Inza M.V., Pomponio M.F., Fernández P., Acuña C.V., Zelener N., Fornés L., Hopp H.E., Paniego N.B, & Marcucci Poltri S.N. (2018). De novo transcriptome sequencing and SSR markers development for Cedrela balansae C.DC., a native tree species of northwest Argentina. PLoS ONE, 13(12), e0203768.

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High-fat diet liver transcriptome analysis

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After feeding a HFD for 14 weeks, the liver samples were randomly selected for RNA-seq (n = 3 for each group). Liver tissue samples were homogenized, and total RNA was extracted using the Qiagen miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. An Agilent 2100 Bioanalyzer and a Nanodrop was used for determination of the isolated RNA quantity and quality. cDNA libraries were constructed using an Illumina TruSeq RNA LS Sample Preparation kit v2 (Illumina, San Diego, CA, USA). Final individual cDNA libraries were assessed for fragment size and quality with the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and sequencing libraries were quantified with the ABI StepOnePlus Real-Time PCR System (Thermo Fisher, Waltham, MA, USA). Paired terminal sequencing was performed on an HiSeq 2500 sequencer (Illumina, San Diego, CA, USA). Differential expression analysis was performed according to the gene expression in different sample groups. GO functional analysis, pathway functional analysis, cluster analysis, and gene–gene interaction network analysis were used to analyze the differentially expressed genes. Transcriptomic data were deposited in the Gene Expression Omnibus under accession code GSE193796.

Zhang Y., Yan T., Wang T., Liu X., Hamada K., Sun D., Sun Y., Yang Y., Wang J., Takahashi S., Wang Q., Krausz K.W., Jiang C., Xie C., Yang X, & Gonzalez F.J. (2022). Crosstalk between CYP2E1 and PPARα substrates and agonists modulate adipose browning and obesity. Acta Pharmaceutica Sinica. B, 12(5), 2224-2238.

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Transcriptome analysis of two invasive snails

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P. canaliculata (20–25 mm shell length; 25.23 ± 0.34 g; 10 individuals) and C.cahayensis (20.4–23.2 mm shell length; 22.43 ± 0.46 g; 10 individuals) were collected without the use of chemicals and grown in the Aquatic Invasive Risk Assessment Center, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China. Tissues samples from the foot, muscle, liver, and kidney were rinsed separately with water pretreated by diethyl pyrocarbonate to cleanse the samples and inactivate RNases [32 (link)]. Total RNA of each sample was extracted using a Trizol Kit (Promega) according to the manufacturer’s instructions. RNA quality was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and RNase-free agarose gel electrophoresis, with the total RNA concentration measured using a 2100 Bioanalyzer. Equal amounts of RNA from each sampled tissue were combined for subsequent experiments and RNA purity was assessed at absorbance ratios of OD_260/280 and OD_260/230. RNA integrity was confirmed by 1% agarose gel electrophoresis.

Mu X., Hou G., Song H., Xu P., Luo D., Gu D., Xu M., Luo J., Zhang J, & Hu Y. (2015). Transcriptome analysis between invasive Pomacea canaliculata and indigenous Cipangopaludina cahayensis reveals genomic divergence and diagnostic microsatellite/SSR markers. BMC Genetics, 16(1), 12.

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Comprehensive DNA and RNA Extraction Protocol for PDXs and PDXOs

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Genomic DNA from cells, PDXs and PDX-derived organoids (PDXOs) was purified using DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA, Cat. 69506) according to the manufacturer’s instructions. DNA integrity was determined by 2100 Bioanalyzer (Agilent) and quantified using NanoDrop (Thermo Scientific). One aliquot of high-quality DNA sample (OD260/280 = 1.8–2.0, OD260/230 ≥ 2.0, >1 μg) was used for deep NGS sequencing and WES sequencing. Total RNA from cells, PDXs and PDXOs was purified using RNeasy Mini Kit (QIAGEN, Cat. 74106) according to the manufacturer’s instructions. Integrity of the total RNA was determined by 2100 Bioanalyzer (Agilent) and quantified using NanoDrop (Thermo Scientific). One aliquot of high-quality RNA sample (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN ≥ 8.0, >1 μg) was used for deep NGS sequencing and RNA-seq.

Chen X., Qian W., Song Z., Li Q.X, & Guo S. (2020). Authentication, characterization and contamination detection of cell lines, xenografts and organoids by barcode deep NGS sequencing. NAR Genomics and Bioinformatics, 2(3), lqaa060.

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RNA-Seq Expression Profiling of Mouse Embryonic Skin

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RNA for RNA-Seq expression profiling was collected from flash-frozen dorsal skin from E15.5, 16.5, 17.5, and E18.5 mouse embryos using the Quick-RNA miniprep kit (Zymo Research) or RNAeasy kit (Qiagen) following manual homogenization using the Tissue Squisher (Zymo Research) in lysis buffer. cDNA was synthesized using 1 μg RNA using the Superscript III First Strand Synthesis Kit (Life Technologies/Thermo Fisher Scientific). RNA quality was determined using the 2100 Bioanalyzer (Agilent). One microgram RNA from each sample was used for mRNA enrichment using NEBNext Poly(A) mRNA magnetic isolation module (E7490S, New England Biolabs). RNA-Seq libraries were constructed using the purified mRNA samples with NEBNext Ultra II Directional RNA library Prep Kit for Illumina (E7760S, New England Biolabs). The quality of these RNA-Seq libraries was validated using 2100 Bioanalyzer (Agilent), and sequencing was performed by NUseq Core Facility using the Illumina Hiseq4000 (1 × 50 bp) or the Advanced Genomics Core of the Skin Biology Diseases Resource–based Center at the University of Michigan using the Illumina NovaSeq 6000 system (Illumina).

Godsel L.M., Roth-Carter Q.R., Koetsier J.L., Tsoi L.C., Huffine A.L., Broussard J.A., Fitz G.N., Lloyd S.M., Kweon J., Burks H.E., Hegazy M., Amagai S., Harms P.W., Xing X., Kirma J., Johnson J.L., Urciuoli G., Doglio L.T., Swindell W.R., Awatramani R., Sprecher E., Bao X., Cohen-Barak E., Missero C., Gudjonsson J.E, & Green K.J. (2022). Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor. The Journal of Clinical Investigation, 132(3), e144363.

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