ILC2s, CD4+ T cells, and TH2 cells were sorted directly into TRIzol LS (Thermo Fisher Scientific). Cultured ILC2s, TH2 cells, or gentleMACS-homogenized lung cells were also frozen in TRIzol LS (Thermo Fisher Scientific), and RNA was isolated using the miRNeasy Micro kit (Qiagen). RNA was reverse-transcribed with the SuperScript III kit (Invitrogen). Primers used for SYBR Green PCRs are as follows: Gapdh: 5′-GTGTTCCTACCCCCAATGTGT-3′, 5′-ATTGTCATACCAGGAAATGAGCTT-3′; Il9: 5′-ATGTTGGTGACATACATCCTTGC-3′, 5′-TGACGGTGGATCATCCTTCAG-3′; Il5: 5′-CTCTGTTGACAAGCAATGAGACG-3′, 5′-TCTTCAGTATGTCTAGCCCCTG-3′; Il13: 5′-CCTGGCTCTTGCTTGCCTT-3′, 5′-GGTCTTGTGTGATGTTGCTCA-3′; Csf2: 5′-GTCTCTAACGAGTTCTCCTTC A-3′, 5′-CCTTGAGTTTGGTGAAATTGCC-3′; Il6: 5′-AGCCAGAGTCCTTCAGAGA-3′, 5′-TCCTTAGCCACTCCTTCTGT-3′; Rora: 5′-CTTCTCGGTGGTTCTTCTGT-3′, 5′-TTCTGCATTCGTACTGATGTCA-3′; Socs-1: 5′-ATTCCACTCCTACCTCTCCAT-3′, 5′-CAGAAAAATGAAGCCAGAGACC-3′; Tnfaip3: 5′-AGAACCAGAGATTCCATGAAGC-3′, 5′-CTGTGTAGTTCGAGGCATGT-3′; Kit: 5′-CCATAGGACCAGACATCACTT-3′, 5′-CTAGCCAGAGACATCAGGAATG-3′; Pten: 5′ TAATATACATAGCGCCTCTGACTG-3′, 5′-CGGACTGGTGTAATGATTTGTG-3′; Muc5ac: 5′-CAGGACTCTCTGAAATCGTACCA-3′, 5′-AAGGCTCGTACCACAGGGA-3′; Clca3: 5′-CTGTCTTCCTCTTGATCCTCCA-3′, 5′-CGTGGTCTATGGCGATGACG-3′; and Gob5: 5′-CATCGCCATAGACCACGACG-3′, 5′-TTCCAGCTCTCGGGAATCAAA-3′.
Trizol ls
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Canada, Australia, Japan, France, China
TRIzol LS is a guanidinium-based reagent used for the isolation of total RNA from various samples, including liquid samples. It is designed to effectively lyse cells and solubilize cellular components while maintaining the integrity of the extracted RNA.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (150 mentions)
Rneasy mini kit, by Qiagen (53 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (47 mentions)
Direct zol rna miniprep kit, by Zymo Research (38 mentions)
Nanodrop, by Thermo Fisher Scientific (37 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (36 mentions)
Qiaamp viral rna mini kit, by Qiagen (35 mentions)
Trizol reagent, by Thermo Fisher Scientific (28 mentions)
Mirneasy mini kit, by Qiagen (26 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (24 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (22 mentions)
Dnase 1, by Thermo Fisher Scientific (21 mentions)
Turbo dnase, by Thermo Fisher Scientific (21 mentions)
Direct zol rna microprep kit, by Zymo Research (21 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (20 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (19 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (18 mentions)
Nextseq 500, by Illumina (17 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (17 mentions)
Facsaria 2, by BD (15 mentions)
1 037 protocols using trizol ls
1
Gene Expression Analysis of Lung Cells
2
SARS-CoV-2 Viral Load Quantification
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Samples collected for PCR analysis were inactivated in TRIzol™ LS (Thermo Fisher Scientific, Waltham, MA, USA) in a ratio of three parts TRIzol™ LS to one part sample. Inactivated samples were then extracted and eluted with AVE buffer using a QIAamp® Viral RNA Mini Kit (Qiagen, Germantown, MD, USA). Samples were tested for viral copies using two qRT-PCR assays targeting the nucleocapsid (N2) and envelope (E) genes. The RT-PCR reaction used Invitrogen™ SuperScript® One-Step RT-PCR System (Thermo Fisher Scientific) with additional magnesium sulfate (MgSO4) added to a final concentration of 3.0 mM. Specimens were run in triplicate using a 5 µL volume on an Applied Biosystems® 7500 Fast Dx instrument (Thermo Fisher Scientific). The average of the triplicates was multiplied by 200 to obtain genomic equivalents per mL, then multiplied by a dilution factor of four (one part biological sample to three parts TRIzol™ LS) for the final reported value. The genomic equivalents were determined using a standard curve of synthetic RNA of known concentration. The sequences of primers and probes for the N2 and E genes from SARS-CoV-2 that were used in the assays were previously described in [13 (link),22 (link),23 (link)].
3
Sputum-Based Transcriptome Analysis
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Sputum samples were collected over the period of two weeks during spring of 2015. Approximately 2 ml from each patient, were collected into 50 ml BD Falcon™ tubes, mixed with 6 ml of Trizol® LS (ThermoFisher Scientific, MA, USA), stored in Trizol® LS at -70°C, until all samples were ready for subsequent DNA and RNA isolation following the manufacturer’s protocol. Quality and quantity of the extracted DNA and RNA were assessed on Implen’s NanoPhotometer® Pearl (München, Germany), and subsequently visualized by performing 2% agarose gel electrophoresis. RNA integrity was examined on the BioRad Experion system (CA, USA). Sample processing and subsequent sequencing reactions were performed in the same batch, using the same respective kits, by the same technician, and at the same laboratory setting at Health GeneTech Corporation, Taoyuan, Taiwan.
4
Total RNA Extraction from Granulomatous Lesions
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Frozen granulomatous lesions were first macerated with the help of a pestle and pot. To proceed with total RNA extration, TRIzol LS (Thermo Scientific) was used according to the protocol described by Pisu et al., 2020 (link). For the control lung was used uninfected healthy lungs, while for the control yeasts was used the original yeast suspension used in the infection. The control lung and control fungus were mechanical lysed with pestle and pot and zirconium beads, respectively, followed by RNA extraction with TRIzol LS (Thermo Scientific) according to the manufacturer’s protocol. After RNA extraction, the DNA was digested with Rnase-free Dnase (RQ1 Rnase-Free Dnase, Promega Corporation) in the presence of the ribonuclease inhibitor Rnasin® (Promega Corporation). The purification was done with the addition of 1 volume of phenol:chloroform (1:1), followed by 100% ethanol:3M sodium acetate (10:1) RNA precipitation. RNA integrity was verified with a 2100 Bioanalyzer (Agilent) and RNA 6000 Nano Kit (Agilent).
5
Real-Time RT-PCR Analysis of p62 in Adipocytes
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Two‐step SYBR real‐time RT‐PCR was performed. Total RNA was isolated from the cultured undifferentiated and differentiated (on day 4 post‐differentiation) primary adipocytes isolated from WAT and BAT using Trizol‐LS (Invitrogen, Carlsbad, CA, USA, Cat# 10‐296‐010) as described by the manufacturer. One µg of RNA was treated with RNase‐free DNase‐I (Thermo Scientific, Cat# EN0521) re‐purified using Trizol‐LS and further used for cDNA preparation with RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Cat# K1621). The SYBR real‐time RT‐PCR was performed using the Power Up SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA, Cat# A25742) using the Bio‐Rad (CFX Connect Real‐Time PCR) detection system. Ct (cycle threshold) values were analyzed using the comparative Ct(ΔΔCt) method. The amount of target p62 (2‐ΔΔCt) was obtained by normalization to an endogenous reference standard (β‐actin) and relative to undifferentiated p62 levels. The following primers were used: β‐actin fwd: 5′‐GCAGGAGTACGATGAGTCCG‐3′, rev: 5′‐ACGCAGCTCAGTAACAGTCC‐3′; p62 fwd: 5′‐TTGAAGTCTTTGGACCCCCG‐3′, rev: 5′‐CTCGAGTCACAGTGGACCCT‐3′.
6
SYBR RT-PCR Analysis of p62 in Adipocytes
7
Total RNA Extraction from Seminal Plasma
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Total RNA was extracted as described previously with minor modification [12 (link)]. In brief, untreated seminal plasma after thawing was immediately confused with Trizol LS (Invitrogen, Carlsbad, USA) at a higher concentration (0.2ml untreated seminal plasma with 0.8ml Trizol LS) to ensure enough quantity for consequent miRNA cDNA synthesis. All procedures were followed according to the manufacturer's instruction.
The purity of total RNA was checked using an ultraviolet spectrophotometer (Biometra, Göttingen, Germany) at 260 nm and 280 nm, and the RNA concentration was calculated using the OD260.
The purity of total RNA was checked using an ultraviolet spectrophotometer (Biometra, Göttingen, Germany) at 260 nm and 280 nm, and the RNA concentration was calculated using the OD260.
8
Immunoprecipitation of Argonaute Proteins
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Argonaute IPs were carried out using affinity-purified rabbit antibodies pre-bound to Protein A Dynabeads (Fisher Scientific) (5–10 ug of antibody per 100 ul of beads)123 (link). Germline or embryo whole-cell lysates were mixed with Protein A Dynabeads and rotated overnight at 4 °C. Protein A Dynabeads were recovered and washed with high-salt buffer (50 mM Tris–HCl, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate) three times. The beads were resuspended in 250 ul of Proteinase K buffer containing 200 µg/ml Proteinase K and incubated for 1 h at 37 °C. RNA was extracted using Trizol LS (Invitrogen) adapted for small RNA recovery by precipitation using 2 volumes of isopropanol, 15 µg of GlycoBlue (Invitrogen), and 30 min incubation at −80 °C followed by centrifugation at 18,500 × g for 35 min at 4 °C. The RNA was treated with RppH (25 units, New England Biolabs) in Thermopol buffer for 5’ independent cloning124 (link). Both untreated and RppH-treated samples were done for AsALG-1 and AsALG-4 samples. Following RppH treatment, samples were repurified with Trizol LS (Invitrogen) (adopted for small RNAs extraction) and stored at −80 °C.
9
Profiling Circulating miRNAs from Plasma Fractions
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RNA was isolated from plasma, circulating MVs, exosomes, and vesicle‐free supernatant (ie, the remaining supernatant after exosome isolation) by using a TRIzol‐based miRNA isolation protocol. For each patient, 250 μL total plasma was diluted in 750 μL TRIzol LS (Life Technologies) to measure plasma miRNA levels. An additional 250 μL total plasma was centrifuged at 20 000g for 30 minutes at 4°C to pellet circulating MVs.18 (link) The pellet was diluted in 250 μL RNase‐free water and then diluted in 750 μL TRIzol LS to measure MV miRNA levels. Caenorhabditis elegans miR‐39 (cel‐miR‐39; 5 nmol/L; Qiagen) was spiked in TRIzol for normalization of miRNA content, as described previously.8 (link) To increase the yield of small RNAs, the RNA was precipitated in ethanol at −20°C overnight with glycogen (Invitrogen).
To analyze miRNA levels in exosomes and vesicle‐free supernatant, the remaining supernatant after 20 000g centrifugation was collected and centrifuged at 100 000g for 1 hour at 4°C to pellet exosomes.19 (link) The pellet was diluted in 250 μL RNase‐free water and then diluted in 750 μL TRIzol LS to measure exosome miRNA levels. The remaining supernatant after exosome isolation, defined as “vesicle‐free supernatant,” was diluted in 750 μL TRIzol LS and processed, as described.
To analyze miRNA levels in exosomes and vesicle‐free supernatant, the remaining supernatant after 20 000g centrifugation was collected and centrifuged at 100 000g for 1 hour at 4°C to pellet exosomes.19 (link) The pellet was diluted in 250 μL RNase‐free water and then diluted in 750 μL TRIzol LS to measure exosome miRNA levels. The remaining supernatant after exosome isolation, defined as “vesicle‐free supernatant,” was diluted in 750 μL TRIzol LS and processed, as described.
10
Quantifying HCMV UL38 Expression and Viral Titers
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MRC-5 cells were nucleofected with 2 μg of UL38 expression plasmid (kindly provided by Scott S. Terhune, (Terhune et al., 2007 (link))) or mock 24 h before infection followed by transfection of 25μM of FD86 or FDScram (using lipofectamine 3000) at 12 h before infection. Cells were infected with AD169 virus (MOI=0.1) and harvested at 4 dpi to quantify UL38 expression and virus titer. For UL38 expression quantification, cells were lysed in TRIzol LS (cat#10296010, Invitrogen), using 0.75ml TRIzol LS per 1 million cells. RNA was extracted using the Direct-zol RNA extraction kit (cat#R2070T, Zymo Research Inc.), DNAse treated using RNAse-free DNAse I (cat#EN0521, Thermofisher Scientific), reverse transcribed using SuperScript II Reverse transcriptase using oligo d(T) primers (cat#12574026, Thermofisher Scientific), and cDNA was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR green PCR master mix (cat#4309155, Thermofisher Scientific) with primers specific to UL38. For quantification of virus titer, cells were subjected to three freeze-thaws followed by centrifugation at 10,000 rpm for 10 minutes, supernatant was harvested and virus titer was quantified by TCID-50.
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