Normal tissue of the lingual mucosa from the volunteer, and mouse tissues of the submandibular gland, tongue, small intestine, and kidney were used after the collection according to the method described above. Immediately after excision 5 mm square tissue specimens were ground into a paste with a scalpel on a glass plate in ice and dissolved in the RLT buffer of an RNeasy kit (Qiagen, Inc.). We also used squamous cell carcinoma cell lines HSC2, HSC3, and HSC4. The cells were dissolved in RLT buffer at the 80% confluence in one well of a 6‐well plate. Furthermore, the buccal mucous epithelial total RNA was collected from volunteers. Immediately after grinding a swab (Jonson's cotton bud was best; Jonson & Jonson K.K., Tokyo, Japan) against the volunteer's buccal mucosa eight times at places facing the tongue, the swab was immersed and spun in the RLT buffer of a RNeasy kit (Qiagen) in a 1.5 ml Eppendorf tube on ice, and the swab, swollen with solution was strongly squeezed by sterilized tweezers. The total RNA extraction from tissue and cells was assayed with a QIAshredder column and an RNeasy kit (Qiagen). Contaminating genomic DNA was removed using DNAfree (Ambion) only when many non‐specific bands were found on the gel electrophoresis after PCR.
Rneasy kit
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, Spain, Netherlands, Canada, France, Japan, China, Italy, Switzerland, Australia, Sweden, India, Singapore, Denmark, Belgium
The RNeasy kit is a laboratory equipment product that is designed for the extraction and purification of ribonucleic acid (RNA) from various biological samples. It utilizes a silica-membrane-based technology to efficiently capture and isolate RNA molecules.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (695 mentions)
Trizol reagent, by Thermo Fisher Scientific (564 mentions)
Trizol, by Thermo Fisher Scientific (551 mentions)
Iscript cdna synthesis kit, by Bio-Rad (529 mentions)
Nanodrop, by Thermo Fisher Scientific (305 mentions)
Quantitect reverse transcription kit, by Qiagen (294 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (283 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (273 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (226 mentions)
Superscript 3, by Thermo Fisher Scientific (221 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (221 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (218 mentions)
Iq sybr green supermix, by Bio-Rad (191 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (190 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (182 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (155 mentions)
2100 bioanalyzer, by Agilent Technologies (153 mentions)
Lightcycler 480, by Roche (149 mentions)
Sybr green master mix, by Thermo Fisher Scientific (149 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (144 mentions)
11 240 protocols using rneasy kit
1
Total RNA Extraction from Oral Tissues
2
Gene Expression Analysis of PEITC Treatment in Cancer Cells
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SK-BR-3, SK-BR-3 transfected with p53 siRNA or NS siRNA, H1299 and A549 cells were treated with 4 μM PEITC or DMSO as a control for 4 h. RNA was extracted from the cells using a Qiagen RNeasy Kit (Qiagen, Valencia, CA, USA), cDNA was synthesized by using High Capacity RNA to cDNA Kit (Applied Biosystems, Invitrogen, Thermofisher Scientific) and the gene expression level was measured by qRT-PCR using TaqMan gene expression assays (Applied Biosystems, Invitrogen). The gene expression level is normalized with GAPDH, and the average is presented with S.D. from triplicates of repeated experiments. RNA was extracted from the SK-BR-3 xenograft tissues by using Qiagen RNeasy Kit and was processed further for qRT-PCR as described for the SK-BR-3 cells. The gene expression levels were normalized with GAPDH. Fold changes in the gene expression levels were calculated for each tumor from the treated group relative to the tumors from the control group and the average is presented with S.D.
3
RNA Isolation from Fresh Frozen Tumors
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RNA from fresh frozen primary tumors (D1, D2, and D3) was isolated using the Qiagen RNeasy kit (Qiagen, Hilden, Germany). Briefly, the tissue was placed in a 4.5 ml cryotube, and 500 µl of QIAzol (Qiagen) was added before the sample was disrupted using Qiagen TissueRuptor (Qiagen), according to the manufacturer’s recommendations. The sample was centrifuged at 18 400 ×g for 10 min to remove insoluble material before being processed with the Qiagen RNeasy kit with DNase. Samples were purified using the Zymo PCR inhibitor removal kit (Zymo, Irvine, CA). RNA from the pelleted samples (adherent and cultured spheres from D1, D2, and D3) was isolated as described above, except the disruption step using the Qiagen TissueRuptor. RNA concentration and purity were determined using NanoDrop (Wilmington, DE) and Bioanalyzer (Agilent 2100, Agilent, Santa Clara, CA). All nine samples had RNA integrity number (RIN) values above 8 before being analyzed with microarray and PCR [24 (link)].
4
DNA Extraction from Leukocytes
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Pre-operative samples of whole blood were collected in EDTA tubes (BD Vacutainer; BD Diagnostics) and the erythrocytes were immediately treated with cold (4–6°C) ammonium lysis buffer (153 mM NH4Cl, 10 mM KHCO3 and 0.01 mM EDTA adjusted to pH 7.4-7.5) until tranluscent. After lysis and washing in PBS, leukocytes were resuspended in 350 µl of RLT Buffer from the Qiagen RNEasy kit (Qiagen AB) and were subsequently frozen. DNA was extracted using the Qiagen RNEasy kit in a QIAcube (Qiagen AB) according to the manufacturer's protocol. Concentration and purity assessments were performed using a NanoDrop 1000 (Thermo Fisher Scientific, Inc.). The 260 nm/280 nm absorbance ratios were in the range of 1.75-1.85.
5
Quantitative RT-PCR Analysis of Metabolic Enzymes
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Quantitative RT-PCR was used to measure relative expression of metabolic enzymes. Relative expression of all mRNA targets was tested using the TaqMan gene expression system (Applied Biosystems, Foster City, CA, USA). Total RNA was extracted using the TRIzol Plus RNA Purification System (Invitrogen, Carlsbad, CA, USA) or a Qiagen RNeasy Kit (Qiagen, Hilden, Germany). A NanoDrop One (Thermo Scientific, Waltham, MA, USA) instrument was used to test the quantity and concentration of RNA. For arntl silencing and overexpression, cell lysis was performed in plate with a Qiagen RNeasy Kit (Germantown, MD) used for downstream RNA processing according to manufacturer protocols. cDNA was synthesized using the TaqMan Reverse Transcription System and run on an Applied Biosystems 7300 Real-Time PCR instrument. TaqMan probes were as follows: nr1d1 (Mm00520711), RPIA (Mm00485790), transketolase (Mm00447559), arntl (Mm00500226), pyruvate kinase muscle isoform (m1/m2) (Mm00834102), pyruvate kinase liver/red blood cell isoform (Mm00443090), phosphofructokinase (Mm01309576), hexokinase I (Mm00439344), hexokinase II (Mm00443385), aldolase c (Mm01298116), and pdhb (Mm00499323). Samples were denatured at 95 °C for 10 min, followed by 40 cycles of denaturing at 95° for 15 s followed by extension at 60 °C for 1 min.
6
Zebrafish RNA Extraction and Reverse Transcription
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To extract RNA, zebrafish were removed from RNAlater and homogenized in 350 μL of RLT buffer in the Qiagen RNeasy kit (Qiagen) with 1 mm glass beads using the Bullet Blender (Next Advance, Troy, NY). Homogenates were transferred to 1.5 mL Eppendorf tubes and spun in a tabletop centrifuge at maximum speed (21000 x g) for 3 min. The supernatant was transferred to 2 mL Eppendorf tubes and RNA was extracted using the Qiagen RNeasy kit and an automated QIAcube robotic workstation (Qiagen) according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). Nucleic acid purity was determined based on the ratio of absorbance at 260 nm to 280 nm (A260/A280 of 1.8–2.2 was considered acceptable), and the absence of genomic DNA contamination was determined by visualization on an agarose gel stained by SYBR Green nucleic acid gel stain (ThermoFisher Scientific). RNA was reverse transcribed, at a concentration of 1 μg of RNA per 20 μL reaction, using Superscript VILO cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions.
7
Isolation of RNA from Cortical Lysates
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Homogenized cortical lysates that were prepared in ice-cold T-per homogenization buffer, as described above, were added to an equal volume of QIAzol lysis reagent (QIAGEN RNeasy kits). RNA was isolated using QIAGEN RNeasy kit as per the manufacturer’s protocol and quantified using NanoDrop (Thermo Scientific).
8
RNA Extraction and Purification Protocol
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Cytology brushes frozen in RNAlater were defrosted on ice and diluted with sterile 1× PBS. Samples were centrifuged at 18,000g for 20 minutes, and brushes were removed and placed into a Lyse E tube. Pellets were resuspended in RLT/β-mercaptoethanol and added to the Lyse E tube. Samples were agitated in a bead beater (MP Biomedicals FastPrep-24 Classic) for 30 seconds. Samples were centrifuged at 500g for 1 minute and transferred to an Allprep (Qiagen) spin column. RNA and DNA were prepared according to the manufacturer’s instructions. Residual DNA was removed from the purified RNA by incubation with RNase-Free DNase (Promega) for 30 minutes at 37°C. DNase was removed from the preparation via a second RNA cleanup using the Qiagen RNeasy Kit. RNA concentration was determined using NanoDrop (Thermo Fisher Scientific), and RNA quality was assessed using Agilent Pico RNA kit.
For cultured cells, ALI Transwell inserts or organoids were lysed in RLT plus buffer (Qiagen) and RNA purified using the Qiagen RNeasy Kit according to the manufacturer’s instructions. Equal quantities of RNA were reverse-transcribed using the SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific) and amplified using Power SYBR Green PCR master mix (Thermo Fisher Scientific) using the primers listed inSupplemental Table 4 .
For cultured cells, ALI Transwell inserts or organoids were lysed in RLT plus buffer (Qiagen) and RNA purified using the Qiagen RNeasy Kit according to the manufacturer’s instructions. Equal quantities of RNA were reverse-transcribed using the SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific) and amplified using Power SYBR Green PCR master mix (Thermo Fisher Scientific) using the primers listed in
9
Tumor Sample Preparation and DNA/RNA Extraction
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Tumor samples from FAIMoS and Edinburgh were microdissected under a stereomicroscope (Olympus SZ61, Tokyo, Japan) using a sterile needle to ensure a tumor cell content >90% as previously described [30 (link)]. DNA extraction was performed by proteinase K digestion and phenol-chloroform precipitation. Total RNA from FAIMoS samples was extracted from un-microdissected sections using the RNeasy kit (Qiagen, Hamburg, Germany) as previously described [31 (link)]. RNA purity and integrity was assessed using the Agilent Bioanalyzer (Santa Clara, CA, USA), with samples only being analyzed if RNA integrity scores were >5.0. Sample processing and RNA extraction from samples of the Edinburgh cohort has been previously described [6 (link)]. For cell lines, DNA was extracted using the DNeasy blood and tissue kit (Qiagen) and RNA was extracted using the RNeasy kit (Qiagen).
10
Quantifying Gene Expression in VEGFB Mice
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Total RNA was isolated from hearts from 20‐week‐old male C57BL/6 Vegfb+/+ (n = 6), Vegfb+/− (n = 6) and Vegfb−/− (n = 6) mice using TRIzol extraction prior to isolation with the RNeasy Kit (Qiagen). RNA isolation from cultured cells was performed using the Qiagen RNeasy Kit, according to manufacturer's instructions. RNA concentration was determined with a NanoDrop instrument, and RNA was transcribed to cDNA with the iScript cDNA synthesis kit (Bio‐Rad). Quantitative real‐time PCR (qPCR) was performed using 10 ng cDNA and run in triplicates. cDNA was mixed with SYBR Mix and 250 nM primer and H2O to 20 μl. Samples were run in a Rotor‐Gene Q from Qiagen or Corbett Research for 40 cycles (1× 95°C 3 min, 40× 3 s 95°C, 20 s 59°C, 2 s 72°C). Samples were normalized to ribosomal protein L19 (RPL19) expression.
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