RNA was extracted from three adjacent macro-dissected 10µm formalin-fixed paraffin-embedded (FFPE) sections from the baseline block of the patients included in the study. The ROCHE High Pure miRNA isolation kit (Roche, Basel, Switzerland) was used following SOP M027 from The Cancer Genome Atlas (TCGA) Program developed by the Biospecimen Core Resource (BCR) at Nationwide Children's Hospital in Columbus, Ohio. Quantification was done using high sensitivity RNA Qubit assays (Thermo Fisher Scientific, Carlsbad, CA).
Qubit rna assay
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
The Qubit RNA assay is a fluorescence-based quantitation method for measuring RNA concentration in biological samples. It provides a sensitive and accurate measurement of RNA content without the interference of contaminants often present in complex samples. The assay utilizes a fluorescent dye that selectively binds to RNA, allowing for precise quantitation using a Qubit fluorometer instrument.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Turbo dnase, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (3 mentions)
Direct zol kit, by Zymo Research (3 mentions)
Iscript supermix, by Bio-Rad (3 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Rneasy kit protocol, by Qiagen (2 mentions)
Hiseq 4000, by Genewiz (2 mentions)
Qubit dna assay, by Thermo Fisher Scientific (2 mentions)
Lobind tube, by Eppendorf (2 mentions)
Hiseq system, by Illumina (2 mentions)
Tapestation system, by Thermo Fisher Scientific (2 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
High pure mirna isolation kit, by Roche (1 mentions)
High pure ffpe rna isolation kit, by Roche (1 mentions)
33 protocols using qubit rna assay
1
RNA Extraction from FFPE Tissue
2
RNA Extraction from Mouse and FFPE Samples
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For the Oxford mouse cohort, RNA was extracted using the RNeasy Micro Kit from Qiagen (74004) and DNase treatment was performed using the DNA-free kit from Life Technologies (AM1906).
For the ACRCelerate mouse cohort, RNA extraction was performed using a Qiagen RNeasy kit according to the manufacturer’s protocols.
For formalin-fixed paraffin embedded (FFPE) samples from human cohorts, 2–10 5 micron sections were extracted using the High Pure FFPE RNA Isolation kit (Roche Life Sciences, Penzberg, Germany) under RNase free conditions following the manufacturer’s protocol. RNA quantity and quality were assessed using the RNA Qubit Assays (High sensitivity and Broad range, Thermofisher) and by Nanodrop, respectively.
For the ACRCelerate mouse cohort, RNA extraction was performed using a Qiagen RNeasy kit according to the manufacturer’s protocols.
For formalin-fixed paraffin embedded (FFPE) samples from human cohorts, 2–10 5 micron sections were extracted using the High Pure FFPE RNA Isolation kit (Roche Life Sciences, Penzberg, Germany) under RNase free conditions following the manufacturer’s protocol. RNA quantity and quality were assessed using the RNA Qubit Assays (High sensitivity and Broad range, Thermofisher) and by Nanodrop, respectively.
3
Bacterial RNA Extraction and Sequencing
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Total RNA was extracted from the stored RNAprotect solution using an RNAeasy Qiagen kit (Qiagen, Valencia, CA) as indicated by the manufacturer’s instructions, followed by an additional treatment with DNase I (Qiagen, Valencia, CA) to remove traces of DNA. rRNA depletion and mRNA enrichment were performed using an Illumina Ribo-Zero rRNA removal kit (catalog no. MRZ116C) according to the instructions of the manufacturer (Illumina, San Diego, CA). RNA concentrations were measured using an RNA Qubit assay (Invitrogen, Burlington, Ontario, Canada), and RNA integrity was evaluated using a Bioanalyzer RNA Pico assay (Agilent Technologies, Santa Clara, CA) (18 (link)). cDNA libraries were constructed using a ScriptSeq Complete kit for bacteria (Epicentre, Madison, WI). cDNA was then purified using an Agencourt AMPure XP system (Beckman Coulter, Beverly, MA), and the second cDNA was generated by adding the Illumina adapters as the forward primer and a ScriptSeq index primer as the reverse primer. The resulting libraries were purified using an AMPure XP system (Beckman Coulter, Beverly, MA), quantified with the DNA Qubit assay, and evaluated using the Bioanalyzer sensitivity DNA assay (Agilent Technologies, Santa Clara, CA). One hundred single reads were generated using the Illumina HiSeq 2000 platform at the National Center for Genome Research in Santa Fe, NM.
4
Sodium Arsenite Stress Response in HeLa Cells
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HeLa cells (ATCC CCL-2) were cultured at 37°C, 5% CO2, 90% humidity in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Cat # 11965–092) supplemented with 10% heat-inactivated fetal bovine serum (GeminiBio #100–106), 2 mM L-Glutamine. The cells were tested for mycoplasma contamination using universal mycoplasma detection kit (ATCC, Cat # 30–1012K). Cells were treated with 500 μM of sodium arsenite (Sigma, Cat # S7400-100G) for 60 minutes and total RNA was isolated using TRIzol reagent (Invitrogen, Cat# 15596-018) following the manufacturer’s recommendation. RNA concentration was quantified using a High Sensitivity (HS) RNA qubit assay (Invitrogen, Cat # Q32852) and Nanodrop ND-1000 (ThermoFisher). RNA integrity was assessed on a 2100 Bioanalyzer (Agilent Technologies) or Qubit™ RNA IQ assay (ThermoFisher).
5
Detailed Brain Tissue RNA Extraction
6
Testes RNA Extraction and Sequencing
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Testes were harvested and homogenized in Trizol reagent (Thermo Fisher) and stored at −80°C prior to processing. Total RNA was extracted from the aqueous phase, mixed with ethanol and purified using the RNeasy kit protocols and reagents (Qiagen). RNA was quantified using the Qubit RNA assay (Thermo Fisher) and 400–500 ng total RNA per sample was used in stranded mRNA-seq library preparation (KAPA Biosystems, KK8481) for Illumina sequencing. Libraries were pooled and sequenced with 2×150 cycles paired-end to an average depth of 19.4 million reads per sample on a HiSeq 4000 by Genewiz (South Plainfield, NJ, USA).
7
Strand-Specific Transcriptome Sequencing and Analysis
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Total RNA was quantified and qualified using a Qubit RNA Assay (Thermo Fisher Scientific, Inc.) and TapeStation RNA ScreenTape (Agilent Technologies, Inc.). cDNA synthesis followed by transcriptome libraries were performed using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, Inc.), where dUTP was incorporated during second strand cDNA synthesis, instead of dTTP, which blocks PCR amplification against the second strand templates, enabling strand-specific library preparation. The resulting transcriptome sequencing libraries were quantified by Qubit DNA Assay (Thermo Fisher Scientific, Inc.) and their fragment size distributions were confirmed by TapeStation D1000 ScreenTape (Agilent). The transcriptome libraries established were loaded onto a next-generation sequencing platform, HiSeq 4000 or equivalent (Illumina, Inc.). Sequencing was performed according to the manufacturer's instructions. Image analysis and base calling were performed using software on the HiSeq instrument. GOSeq (27 (link)) and TopGO software (https://bioconductor.org/packages/release/bioc/html/topGO.html ) were utilized in the Gene Ontology (GO) enrichment analysis for differentially expressed genes. All processes were conducted at Azenta Life Sciences (formerly GENEWIZ).
8
RNA Isolation and Quantification for Gene Expression
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RNA was isolated from triplicate mid-log cultures for each strain using standard techniques. Briefly, cells from each culture were harvested by centrifugation, resuspended in 1 mL of TRIzol (Thermo Fisher), and disrupted by bead-beating. Chloroform was added to 25% of the total volume and total RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research). Samples were then treated with TURBO DNase (Ambion) for 1 hour to remove residual contaminating genomic DNA, followed by clean-up with RNA Clean & Concentrator columns (Zymo Research). RNA was quantified by Qubit RNA Assay (Thermo Fisher) and 25 ng of RNA was used as input in the NanoString nCounter assay (NanoString Technologies) with custom-designed probes to quantify gene expression. Target sequences are listed in S9 Table . Data were analyzed with nSolver version 4 (NanoString Technologies); raw NanoString counts were normalized to internal positive controls to correct for technical variation between assays, and normalized to a housekeeping gene (sigA) to correct for variation in RNA input amount. Background counts from no-input samples were subtracted.
9
EvaGreen and Probe dPCR Efficiency
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Evagreen and probe dPCR efficiency test: A single dilution series was performed for each transcript for the 10 fg and 1 fg variability test. Samples were diluted to ˜10 ng/uL in water and measured again with Qubit RNA Assay (ThermoFisher Scientific). From this working stock, aliquots of 1 ng/uL were prepared for all transcripts. A series of 1 in 10 dilutions in a final volume of 20 u L was performed until desired concentrations were reached. All dilutions were performed in Lo-Bind tubes (Eppendorf).
5 dilution replicates: A separate dilution series comprising five replicates per transcript, beginning with five aliquots at 1 ng/uL, was performed for the EvaGreen and probe efficiency tests as outlined above. Dilution 1 for each of these transcripts was also used for the 100 ag variability test. Dilutions were prepared on the same day as each of the studies were conducted.
5 dilution replicates: A separate dilution series comprising five replicates per transcript, beginning with five aliquots at 1 ng/uL, was performed for the EvaGreen and probe efficiency tests as outlined above. Dilution 1 for each of these transcripts was also used for the 100 ag variability test. Dilutions were prepared on the same day as each of the studies were conducted.
10
RNA-seq Analysis of Homo sapiens Transcriptome
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