RNA was extracted from nectaries by mechanical disruption with a microcentrifuge pestle, and using the RNAqueous®-Micro RNA isolation kit (Ambion, Austin, TX) with Plant RNA Isolation Aid (Ambion, Austin, TX). Agarose gel electrophoresis and UV spectrophotometry were used to assess RNA quality of all samples prior to submission to the University of Minnesota Genomics Center for barcoded library creation and Illumina HiSeq 2500 sequencing. Six TruSeq RNA v2 libraries were created from ~500 ng of total RNA (triplicate samples for immature and mature nectaries) and sequenced via 50 bp, paired-end runs on the HiSeq 2500 using Rapid chemistry. All libraries were pooled and sequenced across two lanes to achieve the equivalent of one lane output. This generated ~368 M total reads (~184 M paired-end reads) and the average quality scores were above Q30.
Rnaqueous micro rna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RNAqueous-Micro RNA Isolation Kit is a product designed for the isolation of micro RNA (miRNA) from small samples. It provides a fast and efficient method for extracting miRNA from various sample types, including cells, tissues, and body fluids.
Automatically generated - may contain errors
Lab products found in correlation
Retroscript reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Hiseq 2500 system, by Illumina (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Random hexamer primer, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr system, by Thermo Fisher Scientific (3 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
7500 real time pcr system machine and, by Bio-Rad (3 mentions)
C1000 touch thermal cycler, by Bio-Rad (3 mentions)
Collagenase, by Thermo Fisher Scientific (2 mentions)
Cd31 fitc, by Thermo Fisher Scientific (2 mentions)
Facsaria 2 cytometer, by BD (2 mentions)
Trypsin edta 0.05, by Thermo Fisher Scientific (2 mentions)
Sca 1 apc clone d7, by Thermo Fisher Scientific (2 mentions)
Ter119 fitc clone ter 119, by Thermo Fisher Scientific (2 mentions)
Cd49f pe clone ebiogoh3, by Thermo Fisher Scientific (2 mentions)
Clone 390, by Thermo Fisher Scientific (2 mentions)
Cd45 fitc clone 30 f11, by Thermo Fisher Scientific (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
31 protocols using rnaqueous micro rna isolation kit
1
Transcriptome Analysis of Nectary Development
2
Prostate Cell Isolation and RNA Extraction
3
RNA Isolation and RT-qPCR Analysis Protocol
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Total RNA was isolated from cells using TRIZOL® Reagent (Invitrogen, Carlsbad, CA, USA) or RNAqueous Micro RNA isolation kit (Ambion® Life Technologies, USA). The RNA isolated by both the methods was treated with DNase (Ambion® Life Technologies, USA) to remove contaminating genomic DNA. cDNA was synthesized with the High capacity cDNA Reverse transcription kit from Applied Biosystems (Foster City, CA, USA). Real-time qPCR was performed by SYBR Green technology (Takyon Low ROX SYBR mix, Eurogentec) and Stratagene MxPro 3000 instrument (Agilent Technologies, USA) in cDNA from isolated samples. Primers used are given in S1 Table . The relative fold change in expression of gene of interest in samples with respect to calibrator was calculated using the formula 2-ΔΔCt.
4
FACS-based Single-Cell Transcriptomics
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Mouse α- and δ-cells were FACsorted from GLU-Venus and Sst-CrexEYFP derived islets, respectively, while β-cells were co-isolated based on their bigger size identified by the forward and side scatter signal, as described previously [14] (link). RNA was isolated and processed using the Ovation Rapid DR Library System (NuGEN) and sequenced using an Illumina HiSeq 2500 system at the Genomics Core Facility, Cancer Research UK Cambridge Institute (Cambridge, UK), as described previously [14] (link).
Single cell suspensions from the small intestine of GIP-Venus or Glu-Venus mice were prepared and separated into fluorescence-positive and fluorescence-negative pools of cells using fluorescence-activated cell sorting (FACS) as described [15] (link). The cells were sorted directly into lysis buffer (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) and the RNA was purified using the RNAqueous-Micro RNA isolation kit (Ambion, Thermo Fisher Scientific, Waltham, MA, USA). cDNA was prepared using Superscript III (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The expression of 379 G protein-coupled receptors was tested using custom-designed 384 well qPCR plates from Bar Harbor BioTechnology. Primer target sequences have been published before [16] (link). A genomic DNA sample was used as a calibrator and relative copy numbers were calculated as described [16] (link).
Single cell suspensions from the small intestine of GIP-Venus or Glu-Venus mice were prepared and separated into fluorescence-positive and fluorescence-negative pools of cells using fluorescence-activated cell sorting (FACS) as described [15] (link). The cells were sorted directly into lysis buffer (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) and the RNA was purified using the RNAqueous-Micro RNA isolation kit (Ambion, Thermo Fisher Scientific, Waltham, MA, USA). cDNA was prepared using Superscript III (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The expression of 379 G protein-coupled receptors was tested using custom-designed 384 well qPCR plates from Bar Harbor BioTechnology. Primer target sequences have been published before [16] (link). A genomic DNA sample was used as a calibrator and relative copy numbers were calculated as described [16] (link).
5
Bladder RNA Extraction via LCM
6
Laser Capture Microdissection of Hippocampal Neurons
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Laser capture microdissection (LCM) was performed using a PixCell IIe laser capture microscope with an infrared diode laser (Life Technologies, CA). Using serial sections immediately adjacent (before and after) to the miR-15b ISH/FJC labeled slides, we identified dying, Fluoro-Jade positive neurons and adjacent surviving, Fluoro-Jade negative neurons and laser captured them from the ipsilateral (directly under the injury site) rat hippocampus. Contamination from adjacent cells was minimized by using the smallest laser spot size (7.5 micron) and a power setting range of 75–100 mW with pulse duration of 0.45–0.85 ms, the last two settings adjusted as necessary for optimum capture. Surviving and dying neurons were separately captured on thermoplastic films of CapSure Macro LCM Caps (Life Technologies, CA). Caps were then secured on 0.5 mL tubes with 100 μL lysis solution from the RNAqueous-MicroRNA isolation kit (Ambion, Austin, TX) and vortexed for 15 sec, stored at −20°C, and vortexed 30 sec before the RNA isolation procedure.
7
Organ of Corti RNA Expression Analysis
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Mice were euthanized by CO2 inhalation, then decapitated, ears removed by gross dissection, placed in ice-cold HBSS, and then microdissected for the organ of Corti. Organ of Corti RNA was isolated using the RNAqueous-Micro RNA Isolation Kit (Ambion, Austin, TX, USA). Isolated RNA was treated with DNase I before cDNA synthesis. cDNA was generated using Superscript First-Strand cDNA Synthesis system for RT-PCR (Invitrogen) with random primers.
Relative expression levels were assayed utilizing TaqMan Gene Expression Master Mix and TaqMan probes (Applied Biosystems, Foster City, CA, USA) for Cflar, Bcl2l1, Bcl2l11, Cdkn1a, Hspa1a, 18S, GAPDH, and Actb. Reactions were run in triplicate in an Eppendorf Realplex2 Mastercycler System (Hauppauge, NY, USA). The level of 18S, GAPDH, and Actb were used as internal controls and were run as a multiplex reaction with each assayed gene. The difference in CT between the assayed gene and 18S, GAPDH, and Actb for any given sample was defined as ΔCT(X). The difference in ΔCT(x) between two samples was defined as ΔΔCT(X), which represents a relative difference in expression of the assayed gene. The fold change of the assayed gene relative to 18S, GAPDH, and Actb was defined as 2−ΔΔCT.41 (link) DataAssist software (Applied Biosystems) was used for statistical analysis and to confirm ΔCT(X) calculation.
Relative expression levels were assayed utilizing TaqMan Gene Expression Master Mix and TaqMan probes (Applied Biosystems, Foster City, CA, USA) for Cflar, Bcl2l1, Bcl2l11, Cdkn1a, Hspa1a, 18S, GAPDH, and Actb. Reactions were run in triplicate in an Eppendorf Realplex2 Mastercycler System (Hauppauge, NY, USA). The level of 18S, GAPDH, and Actb were used as internal controls and were run as a multiplex reaction with each assayed gene. The difference in CT between the assayed gene and 18S, GAPDH, and Actb for any given sample was defined as ΔCT(X). The difference in ΔCT(x) between two samples was defined as ΔΔCT(X), which represents a relative difference in expression of the assayed gene. The fold change of the assayed gene relative to 18S, GAPDH, and Actb was defined as 2−ΔΔCT.41 (link) DataAssist software (Applied Biosystems) was used for statistical analysis and to confirm ΔCT(X) calculation.
8
Quantitative RT-PCR Analysis of Mouse Heart
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The total mouse heart RNA was isolated with an RNeasy mini kit (QIAGEN) or RNAqueous Micro RNA isolation kit (Ambion) following the manufacturer's instructions. RNA was reverse-transcribed using a High-Capacity cDNA Archive Kit (Applied Biosystems). qRT-PCRs were carried out in triplicate in a LightCycler 480 System (Roche) using either probe/primer sets or SYBR Green I (see primer list). Relative quantitation of PCR products used the ΔΔCt method relative to two validated reference genes (Tbp1 and Polr2a). All probes and primers were from Roche or Thermo Fisher.
9
Quantitative RT-PCR Analysis of Cochlear Gene Expression
10
Prostate Cell Isolation and RNA Extraction
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Prostate lobes were dissociated and stained for FACS based on published methods20 (link). Briefly, prostate lobes were dissected, minced, and incubated at 37°C in 10%FBS/DMEM containing 1 mg/ml collagenase (Gibco, cat. no. 17018–029). Further dissociation to a single cell suspension was achieved by incubation in Trypsin/0.05% EDTA (Invitrogen cat. no. 25300) for 5 minutes at 37°C, followed by serially passing through 18-G and 20-G needles and filtering through a nylon mesh filter with a 40 μm pore size. Cells were stained for 20 minutes at 4°C. Antibodies used are: Sca-1-APC (clone D7; eBioscience, cat. no. 17–5981–82), 1:500; Ter119-FITC (clone TER-119; eBioscience, cat. no. 11–5921–85), 1:250; CD31-FITC (clone 390; eBioscience, cat. no. 11–0311–85), 1:250; CD45-FITC (clone 30-F11; eBioscience, cat. no. 11–0451–85), 1:250; CD49f-PE (clone eBioGoH3; eBioscience, cat. no. 12–0495–83) 1:333. Cells were sorted using a FACS AriaII cytometer (BD Biosciences), and analysis of flow cytometry data was performed using FlowJo Software (Treestar). Following FACS, isolated cells were centrifuged at 500g for 5 minutes. The pellet washed in PBS, homogenized in lysis buffer from the RNAqueous-Micro RNA Isolation Kit (Ambion AM1931), the mirVana miRNA Isolation Kit (Ambion AM1560) and RNA extracted following kit instructions.
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