RNA was isolated from flash frozen tissue using an RNeasy Kit (Qiagen), and 100 ng of RNA was processed by the Molecular Biology Core at Dana-Farber Cancer Center, Boston, MA, USA. Samples were assessed for quality and concentration (Agilent Bioanalyzer RNA Nano or Pico chips); smear analyzed (Agilent 2100 Expert software) to quantify the percentage of RNA fragments greater than 300 nt; capture and reporter Code sets added; samples hybridize at 65°C for 16 hours, washed and loaded onto a custom-made cartridge (XT-GXA-P1CS-096) using the nCounter Analysis System Prep Station. The cartridge was scanned using the nCounter Digital Analyzer at the maximum resolution of 1150 FOV. Data analysis was completed using nSolver software. Gene expression normalization was performed using the housekeeper genes, tbp and tubb5.
Bioanalyzer rna nano
Manufactured by Agilent Technologies
Sourced in Netherlands, United States
The Bioanalyzer RNA Nano is a lab equipment designed for the analysis of RNA samples. It uses microfluidic technology to assess the quality and quantity of RNA in a small sample. The Bioanalyzer RNA Nano provides detailed information about the size, concentration, and integrity of RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Biomek 400 automated workstation, by Beckman Coulter (2 mentions)
Bioanalyzer dna hs reagents, by Roche (2 mentions)
Kapa qpcr, by Roche (2 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Agilent 2100 expert software, by Agilent Technologies (1 mentions)
Pico chips, by Agilent Technologies (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
5500 solid fragment library core kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin total rna isolation kit, by Macherey-Nagel (1 mentions)
Qubit rna broad range, by Thermo Fisher Scientific (1 mentions)
Solid 5500 wildfire, by Thermo Fisher Scientific (1 mentions)
Ovation rna seq system, by Tecan (1 mentions)
Innuprep plant rna kit, by Analytik Jena (1 mentions)
Rna seq, by Illumina (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra 2 rna library prep kit for, by Illumina (1 mentions)
5 protocols using bioanalyzer rna nano
1
nCounter Gene Expression Analysis Protocol
2
Alligator Transcriptome RNA Sequencing
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Tissue samples were collected and homogenized with a homogenizer (Ultra-Turrax, IKA, DE) in lysis buffer RA1 of the NucleoSpin total RNA isolation kit (RNA II, Macherey-Nagel, DE). RNA isolation was performed following manufacturer’s protocol. RNA purity, concentration and integrity were determined by respectively Nanodrop (ND-1000, Isogen Life Science, NL), Qubit RNA Broad-Range (2.0, Life Technologies), and Bioanalyzer RNA Nano (2100, Agilent Technologies). Amplified double stranded cDNA was generated using the Ovation RNA-Seq system (V2, Nugen) following manufacturer’s protocol with 32 ng RNA input per sample. cDNA purity, concentration and size distribution were determined by Nanodrop, Qubit DNA HS and Bioanalyzer DNA 1000. Libraries were made using the 5500 SOLiD Fragment Library Core kit (Life technologies) and size distribution was determined by Bioanalyzer DNA 1000. RNA sequencing was performed on the SOLiD 5500 Wildfire (Life technologies). Sequence tags were mapped with Lifescope on the AllMis1 Aug. 2012 contig assembly of the American alligator genome.
3
Radicle RNA Extraction for Sequencing
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The distal halves (tips) of radicles were used for RNA extraction. For the initial Illumina RNA-Seq, one radicle of each genotype was subjected to total RNA isolation. For replicate datasets, three frozen radicles of one genotype were pooled prior to RNA extraction. Total RNA was isolated using the innuPREP Plant RNA Kit (Analytik Jena, Jena, Germany) following the manufacturer’s instructions. A DNase I (Fermentas, St. Leon-Roth, Germany) digestion step on the column was included to remove genomic DNA. RNA concentration and integrity were assessed with Bioanalyzer RNA Nano or Pico chips (Agilent, Santa Clara, USA).
For qRT-PCRs with leaf and shoot tip material, samples were transferred to liquid nitrogen, disrupted with a mortar and pestle and RNA was isolated using the innuPREP Plant RNA Kit (Analytik Jena, Jena, Germany) following the manufacturer’s instructions.
For qRT-PCRs with leaf and shoot tip material, samples were transferred to liquid nitrogen, disrupted with a mortar and pestle and RNA was isolated using the innuPREP Plant RNA Kit (Analytik Jena, Jena, Germany) following the manufacturer’s instructions.
4
Isolation and Characterization of Mutant mkaku41 Transcriptome
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Segregating wildtype and mutant mkaku41 plants were grown in the greenhouse. From 2 week-old plants, fourth leaves were harvested and from 6 to 8 week old plants, mid-prophase meiotic-staged male flowers were harvested. The tissues were immediately stored in liquid nitrogen. RNA was isolated from three biological replicates for each genotype using Qiagen RNeasy Plant mini kit per manufacturer’s instructions. Integrity of the RNA was tested using the Bioanalyzer (Agilent) system. For library preparation, sample input was 400 ng total RNA (determined by Qubit RNA HS reagents, Thermo) with RIN > 7 (Bioanalyzer RNA Nano, Agilent). Libraries were prepared with the Biomek 400 Automated Workstation (Beckman Coulter), using the NEBNEXT Ultra II RNA Library Prep kit for Illumina (New England Biolabs) according to manufacturer’s instructions, with an RNA fragmentation time of 15 min, a 1/10th dilution of NEB adaptor and 11 cycles of PCR amplification with dual-indexing primers. Amplified libraries were initially quantified by Qubit DNA HS reagents, checked for size and artifacts using Bioanalyzer DNA HS reagents, and KAPA qPCR (KAPA Biosystems) was used to determine molar quantities of each library. Individual libraries were diluted and pooled equimolar, and the pool was again checked by Bioanalyzer and KAPA qPCR before submission for sequencing.
5
Transcriptome Analysis of Meiotic Mutant Plants
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Segregating wildtype and mutant mkaku41 plants were grown in the greenhouse. From two week-old plants, fourth leaves were harvested and from 6-8 week old plants, midprophase meiotic-staged male flowers were harvested. The tissues were immediately stored in liquid nitrogen. RNA was isolated from three biological replicates for each genotype using Qiagen RNeasy Plant mini kit per manufacturer's instructions. Integrity of the RNA was tested using the Bioanalyzer (Agilent) system. For library preparation, sample input was 400 ng total RNA (determined by Qubit RNA HS reagents, Thermo) with RIN >7 (Bioanalyzer RNA Nano, Agilent). Libraries were prepared with the Biomek 400 Automated Workstation (Beckman Coulter), using the NEBNEXT Ultra II RNA Library Prep kit for Illumina (New England Biolabs) according to manufacturer's instructions, with an RNA fragmentation time of 15 minutes, a 1/10 th dilution of NEB adaptor and 11 cycles of PCR amplification with dual-indexing primers. Amplified libraries were initially quantified by Qubit DNA HS reagents, checked for size and artifacts using Bioanalyzer DNA HS reagents, and KAPA qPCR (KAPA Biosystems) was used to determine molar quantities of each library. Individual libraries were diluted and pooled equimolar, and the pool was again checked by Bioanalyzer and KAPA qPCR before submission for sequencing.
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