For RNA extraction from mouse tissue, freshly collected tissue samples were flash-frozen and transferred to RNAlater-ICE Frozen Tissue Transition Solution (Invitrogen). After soaking overnight at −20°C, the tissue samples were homogenized in vials containing 1.4 mm ceramic beads (Fisherbrand) and 400 µl RLT buffer (Qiagen) using a bead mill (Bead Ruptor 24, Biotage). 200 µl of the tissue homogenate was mixed with 1 ml of TRI Reagent (Invitrogen). For extraction of RNA from cultured cells, the cell pellet was directly resuspended in TRI Reagent. Total RNA extraction was performed according to the manufacturer’s protocol. The resulting RNA was treated with 2U DNaseI enzyme (NEB) for 30 min at 37°C, followed by acidic phenol extraction and isopropanol precipitation. To generate cDNA, about 200 ng of RNA was used in a reverse transcription reaction with SuperScript IV VILO Master Mix (Invitrogen). To measure the relative expression levels of mRNAs by RT-qPCR, FastStart Universal SYBR Green Master (ROX) from Roche was used together with gene-specific primers listed in Supplementary file 1 . GAPDH/Gapdh was used as housekeeping gene.
Rnalater ice frozen tissue transition solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
RNAlater™-ICE Frozen Tissue Transition Solution is a reagent designed to stabilize RNA in tissues during the transition from fresh to frozen samples. It helps preserve the integrity of RNA molecules in tissue specimens prior to RNA extraction and analysis.
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Lab products found in correlation
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Tri reagent solution, by Thermo Fisher Scientific (2 mentions)
Invitrogen glycoblue coprecipitant, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
1.4 mm ceramic beads, by Thermo Fisher Scientific (1 mentions)
Bead ruptor 24, by Biotage (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 4 vilo master mix, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Thermo scientific nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Nsolver analysis software, by NanoString (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (1 mentions)
Smarter universal low input rna kit for sequencing, by Takara Bio (1 mentions)
15 protocols using rnalater ice frozen tissue transition solution
1
RNA Extraction and qRT-PCR from Mouse Tissues
2
RNA Extraction from Renal Cortices
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Renal cortices were removed and snap frozen in liquid nitrogen. Frozen tissues were placed in pre-chilled (−80 °C) RNAlater®-ICE Frozen Tissue Transition Solution (Invitrogen by Thermo Fisher Scientific) and stored at −80 °C until samples were processed for RNA isolation. Total RNA was isolated from the tissues using the mirVana™ miRNA Isolation Kit (Invitrogen by Thermo Fisher Scientific) following the manufacturer’s recommended protocol. The integrity of the RNA samples was evaluated using a Thermo Scientific™ NanoDrop™ 2000 spectrophotometer, (Thermo Fisher Scientific) and by examining 28S and 18S ribosomal RNA bands using denaturing agarose gel electrophoresis and ethidium bromide staining.
3
Quantifying miRNA Expression in Frozen Muscle
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A piece of frozen gastrocnemius muscle was taken from the IHC mount and placed into RNAlater‐ICE Frozen Tissue Transition Solution (Invitrogen) overnight at −20°C. The following morning, residual OCT was gently removed from each piece of tissue. RNA was then isolated using the Qiagen miRNeasy Mini Kit (Qiagen, Germantown, MD, USA). Total RNA concentration was determined using a NanoDrop (ThermoFisher), and then, RNA quality was assessed using an Agilent 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA, USA). MiRNA abundance was then quantified using quantitative single molecule direct digital detection (mouse nCounter miRNA Expression Panel, NanoString, Seattle, WA, USA) and analysed using nSolver Analysis Software (NanoString). The nCounter panel multiplexes 577 mouse miRNAs as well as 6 positive controls, 8 negative controls, 3 ligation positive and 3 negative controls, 5 mRNA reference controls, and 5 spike‐in controls to ensure maximum accuracy. Raw read counts were normalized to the geomentric mean of the positive internal controls for each sample, then normalized to the geometric mean of the top 50 most abundant miRNAs (the geometric mean for the top 50 miRNAs was not significantly different between groups, analysed via one‐way ANOVA). The minimum threshold was set at 50 copies. All miRNAs that fell below this threshold were excluded from the analysis.
4
Transcriptomic Profiling of Ovarian Cancer
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RNA-seq analysis was performed on primary ovarian cancer solid tumor, primary ascites cells, non-huPDX solid tumor, and non-huPDX ascites cells for select patients. Formalin-fixed paraffin-embedded (FFPE) sections of matched patient solid tumor were obtained through the UNM Human Tissue Repository. For patient solid tumor samples, RNA isolation was performed by the UNMCCC Analytical and Translational Genomics (ATG) Shared Resource, as described previously (23, 24 (link)). Briefly, total RNA was isolated from slide-mounted FFPE sections using the RNeasy FFPE kit (Qiagen). For PDX samples, snap-frozen solid tumor was incubated in RNAlater-ICE Frozen Tissue Transition Solution (Invitrogen) for 24 hours at −20°C prior to DNA/RNA extraction with AllPrep DNA/RNA Mini Kit (Qiagen). Cryopreserved patient ascites cells and PDX cancer spheroids were thawed and rinsed in serum-free RPMI before RNA/DNA extraction. cDNA synthesis and library preparation were performed in the UNMCCC ATG Shared Resource using the SMARTer Universal Low Input RNA Kit for Sequencing (Clontech) and the Ion Plus Fragment Library Kit (Life Technologies) as described previously (24 (link)).
5
Spleen RNA Extraction Protocol
6
Hippocampal Transcriptome Analysis in Rodents
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As soon as behavioral tests were completed (on the last day of 4 consecutive weeks for CVS), animals were immediately euthanized in about 10 min on the same day and, then, tissues were dissected and collected on ice and frozen quickly. Total RNA from hippocampus of the rodents were utilized for building library constructions and, then, running the Ilumina whole transcriptome RNA sequencer. The frozen brain was placed in the RNAlater®-ICE Frozen Tissue Transition Solution (Thermo Fisher Scientific, Waltham, MA) at −20°C for overnight and dissected hippocampus, and total RNA was extracted from the whole hippocampus using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to instructions provided in the kit. RNA samples were shipped overnight to the UCLA Biosequencing core where the Illumina RNA sequencer was used for whole transcriptome RNA-sequencing. Samples were multiplexed, tagged with standard Illumina tags and sequenced using Illumina next-gen RNA-Sequencing by trained technicians.
7
RNA Extraction and Sequencing from Cell Lines and Tissues
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RNA from cell lines was obtained by lysing 1-2 million cells using 1 mL of Trizol (Invitrogen) then mixed with 1/5th volume of chloroform and centrifuged at 200×g for 5 min. The aqueous phase was collected and processed using a RNeasy Mini column (Qiagen). cDNA was synthesized from 1 μg of extracted RNA using SuperScript® III Reverse Transcriptase (ThermoFisher Scientific) with oligo(dT), random hexamers, and RNase inhibitor. Mouse brain tissue samples were collected from the cortex, cerebellum and striatum of heterozygous Mbd5+/GT mice and wild-type controls at 8 weeks. Tissues that were previously frozen at −80°C were thawed overnight at −20°C submerged in RNAlater®-ICE Frozen Tissue Transition Solution (ThermoFisher Scientific) enabling easy cutting and extraction of high-quality RNA. RNA from tissues was obtained by lysing in 1 mL of Trizol (Invitrogen) using metallic pellets (Qiagen) and a tissue lyser, then mixed with 1/5th volume of chloroform and centrifuged at 200×g for 5 min. The aqueous phase was collected and mixed with isopropanol and centrifuged to obtain a pellet that was then washed with 75% ethanol and air dried and resuspended in RNAse-free water. Each tissue type was collected on the same day to avoid batch effects. The RNAseq library was prepared as previously described [29 (link)].
8
Extraction of High-Quality RNA from Snap-Frozen Tissues
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Snap-frozen tissues were first treated with RNAlater™-ICE Frozen Tissue Transition Solution (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions, to allow handling of tissue without RNA degradation occurring due to thawing of sample. Tissue sections were placed in 1 mL TRI Reagent® Solution (ThermoFisher, Waltham, MA, USA) supplemented with the addition of 10 mM MgCl2 to aid recovery of microRNAs (Kim et al. 2012 (link)). Samples were completely homogenised in a bead mill (Retsch Technology GmbH, Haan, Germany) at a frequency of 30 cycles per second for 15 min. Phase separation was carried out using chloroform. Total RNA was precipitated from the aqueous phase by means of an overnight incubation at − 20 °C with isopropanol. A total of 1.2 μl Invitrogen™ GlycoBlue™ Coprecipitant (ThermoFisher, Waltham, MA, USA) was added prior to incubation to aid pellet recovery. RNA pellets were ethanol-washed twice and re-suspended in 1× TE buffer, pH 8.0. RNA quality and concentration were assessed by NanoDrop spectrophotometry (NanoDrop, Wilmington, DE, USA).
9
Profiling T Cell Receptor Repertoire
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Freshly biopsied specimens were immediately frozen at −80 °C. Thirty samples obtained at baseline and on-treatment from 15 patients were analyzed. Total RNA was extracted using RNAlater-ICE Frozen Tissue Transition Solution (Thermo Fisher Scientific) according to the instructions provided by the manufacturer. After the PCR step was performed using a primer set that enriched the V, J, and C regions of hTRA (human TCR alpha), the PCR products were measured using a Qubit Fluorometer (Thermo Fisher Scientific) and checked by electrophoresis for quality. Eleven of 30 samples were below the required level for analysis and excluded from further analysis. Purified products were sequenced by Mi-Seq (Illumina, San Diego, US), and sequence data were analyzed by Repertoire Genesis version 20180912 software (Repertoire Genesis, Osaka, Japan). According to the number of sequenced unique reads for complementarity-determining region three (CDR3) of TRA, the diversity (Shannon-Weaver index H, Inverse Simpson’s index 1/gamma, Pielou’s evenness) or clonality (diversity evenness score [DE50]) of the TCR repertoire in each sample were calculated18 (link).
10
Microrna Profiling of Rat and Human Lingual Nerve
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Rat lingual nerve tissue was disrupted and miRNA isolated using the mirVana miRNA Isolation Kit (ThermoFisher, Paisley, UK) according to the manufacturer’s protocol. Human neuroma tissue miRNA was isolated using the RecoverAll Total Nucleic Acid Isolation Kit (ThermoFisher). As the samples used were frozen and not embedded in paraffin, the deparaffinisation step was omitted from the manufacturer’s protocol. Instead, prior to extraction, human tissue samples were stored in RNAlater-ICE Frozen Tissue Transition Solution (ThermoFisher) overnight at –20°C, to facilitate the thawing of the tissue while preserving RNA quality.
miRNA expression profiles were initially screened using TLDA Rodent miRNA Cards v.3 A and B, and TLDA Human miRNA Cards v.3 A and B, respectively, for rat and human lingual nerve samples. Each card held up to 381 preloaded miRNA probes and endogenous controls (according to the miRBase database,38 there are currently 496 verified rat miRNA sequences and 1,917 verified human miRNA sequences). All samples were reverse-transcribed using the TaqMan miRNA Reverse Transcription kit (ThermoFisher) and preamplified (12 cycles) using TaqMan PreAmp master mix and primers, prior to being loaded into the respective TLDA microfluidic cards following the manufacturer’s protocol.
miRNA expression profiles were initially screened using TLDA Rodent miRNA Cards v.3 A and B, and TLDA Human miRNA Cards v.3 A and B, respectively, for rat and human lingual nerve samples. Each card held up to 381 preloaded miRNA probes and endogenous controls (according to the miRBase database,38 there are currently 496 verified rat miRNA sequences and 1,917 verified human miRNA sequences). All samples were reverse-transcribed using the TaqMan miRNA Reverse Transcription kit (ThermoFisher) and preamplified (12 cycles) using TaqMan PreAmp master mix and primers, prior to being loaded into the respective TLDA microfluidic cards following the manufacturer’s protocol.
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