First telogen HFSCs from control and K15-CrePGR; Adrb2 fl/fl male mice were FACS sorted and collected into TRIzol® LS Reagent (Invitrogen). RNA was isolated with an RNeasy Micro Kit (Qiagen), using a QIAcube according to the manufacturer’s instructions. RNA concentration and RNA integrity were determined by Bioanalyzer (Agilent, Santa Clara, CA) using the RNA 6000 Nano chip. High quality RNA samples with RNA Integrity Number ≥ 8 were used as input for RT-PCR and RNA-sequencing. For Shh, Dhh, and Ihh quantitative real time PCR, newborn pups were used. The mouse back skin was dissected. The skin was incubated with 0.25% Collagenase (Sigma c2674) in Hank’s Balanced Salt Solution (HBSS) at 37°C for 20 – 35 minutes on an orbital shaker. The dermal side was scraped, and cells were collected and incubated for 10 minutes in trypsin-EDTA at 37°C to generate a single cell suspension. The remaining tissue was incubated in trypsin-EDTA at 37°C and scraped again. All cells were collected together and filtered through 70-μm and 40-μm filters. Single cell suspensions were then centrifuged for 8 minutes at 350g at 4°C and re-suspended in TRIzol® LS Reagent (Invitrogen). RNA isolation was performed using ZYMO RESEARCH Direct-Zol RNA Micro-Prep kit (zr2060) according to the manufacturer’s protocol.
Trizol ls reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Canada, United Kingdom, China, Japan, France, Switzerland, Australia
TRIzol LS reagent is a liquid solution used for the isolation and purification of total RNA from various biological samples. It is a mono-phasic solution containing phenol and guanidine isothiocyanate. The reagent facilitates the separation of RNA from DNA and proteins during the RNA extraction process.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (118 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (51 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (50 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (38 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (37 mentions)
Rneasy mini kit, by Qiagen (34 mentions)
Dnase 1, by Thermo Fisher Scientific (33 mentions)
Primescript rt reagent kit, by Takara Bio (31 mentions)
Trizol, by Thermo Fisher Scientific (29 mentions)
Nanodrop, by Thermo Fisher Scientific (29 mentions)
Mirneasy mini kit, by Qiagen (28 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (27 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (26 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (26 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (23 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (19 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (18 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (18 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (17 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (17 mentions)
1 399 protocols using trizol ls reagent
1
FACS Isolation and RNA Extraction of Telogen HFSCs
2
Quantification of EV71 Viral RNA
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Total cellular RNA was isolated with TRIzol reagent according to standard protocols. The following primer sequences were used for qRT-PCR: GAPDH, forward primer 5′-CCCACTCCTCCACCTTTGACG-3′ , reverse primer 5′-CACCACCCTGTTGCTGTAGCCA-3′ , EV71 5′-UTR forward primer 5′-TGAATGCGGCTAATCCCAACT-3′ , and reverse primer 5′-AAGAAACACGGACACCCAAA G- 3′ . qRT-PCR was performed with a QuantiTect SYBR Green RT-PCR kit (Qiagen), and the EV71 and GAPDH transcript levels were determined by ΔΔCT methods.
To determine the amount of purified EV71 virions, viral RNA was extracted from 50 µl of PBS buffer containing EV71 virions by using TRIzol LS reagent (Invitrogen). To determine the amount of EV71 virions in the CsCl fractions, viral RNA was extracted from 125 µl of the CsCl fraction containing EV71 virions with an additional 125 µl of nuclease-free water by TRIzol LS reagent (Invitrogen). A pUC18-EV71AH1 plasmid was used as a standard sample to generate a standard curve ranging from 1011–103 copies/ml. EV71 RNA copies were quantified by using the QuantiTect SYBR Green RT-PCR kit (Qiagen).
To determine the amount of purified EV71 virions, viral RNA was extracted from 50 µl of PBS buffer containing EV71 virions by using TRIzol LS reagent (Invitrogen). To determine the amount of EV71 virions in the CsCl fractions, viral RNA was extracted from 125 µl of the CsCl fraction containing EV71 virions with an additional 125 µl of nuclease-free water by TRIzol LS reagent (Invitrogen). A pUC18-EV71AH1 plasmid was used as a standard sample to generate a standard curve ranging from 1011–103 copies/ml. EV71 RNA copies were quantified by using the QuantiTect SYBR Green RT-PCR kit (Qiagen).
3
Serum RNA Isolation Protocol
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Blood samples were centrifuged at 15000g to separate the sera. All of the samples were collected and stored at −70°C and thawed immediately before assay. Total RNA was isolated from serum using TRIzol LS reagent (Invitrogen, Life Technologies, Paisley, UK) according to manufacturer's instructions with the following modifications: In brief, each 250 μl serum sample was mixed with 750 μl TRIzol LS reagent. After 5 min incubation at room temperature, 200 μl of chloroform was added, followed by 15 sec of shaking and 10 min of incubation at room temperature. The mixture was centrifuged at 12, 000g for 15 min at 4°C in a concentrator (Eppendorf-Netheler-Hinz, Hamburg, Germany). The aqueous layer containing RNA was transferred into a new tube, then RNA was precipitated for 16 hr at −20°C with 0.5 ml isopropyl alcohol and washed with 1 ml of 75% ethanol. Finally, the RNA pellet was dried for 5–10 min at room temperature, dissolved at 60°C in 15 μl of RNase-free water. The RNA concentration was measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific). The final concentrations of RNA ranged from 319∼782 ng/μl. The TRIzol method is described in other studies [53 (link), 54 (link)].
4
RNA Extraction from Organs and Plasma
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Total RNA from organs and plasma was isolated using Cell/Tissue miRNA Purification Kit (Genolution) and TRIzol LS Reagent (Thermo Fisher Scientific), respectively. Frozen organ samples were cut into small pieces in lysis buffer on ice to prevent thawing. RNA extractions from organs were performed according to the manufacturer's instructions. Extracted RNA was and stored at −80 °C. The total RNA concentration and quality were assessed using NanoDrop Lite Spectrophotometer 120 V (Thermo Fischer Scientific, MA, USA) at the absorbance of 260, and 280 nm.
Plasma samples were mixed by TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA), followed by chloroform. Each sample was vortexed for 30 seconds and incubated at RT for 5 minutes. Phase separation was performed by centrifuging the samples for 12 000 × g for 15 minutes at 4 °C. 400 μl of the aqueous phase was transferred to a new tube. 500 μl of isopropanol was added to the aqueous phase and added 5 mg ml−1 glycogen (Invitrogen, San Diego, CA, USA) and 10 μg ml−1 yeast tRNA (Sigma-Aldrich, St. Louis, MO, USA) for RNA precipitation. After precipitation of RNA incubate in −80 °C for 1 hour, centrifuge at 20 000 × g for 30 minutes at 4 °C. The RNA pellet was dissolved with 30 μl of RNase-free water.
Plasma samples were mixed by TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA), followed by chloroform. Each sample was vortexed for 30 seconds and incubated at RT for 5 minutes. Phase separation was performed by centrifuging the samples for 12 000 × g for 15 minutes at 4 °C. 400 μl of the aqueous phase was transferred to a new tube. 500 μl of isopropanol was added to the aqueous phase and added 5 mg ml−1 glycogen (Invitrogen, San Diego, CA, USA) and 10 μg ml−1 yeast tRNA (Sigma-Aldrich, St. Louis, MO, USA) for RNA precipitation. After precipitation of RNA incubate in −80 °C for 1 hour, centrifuge at 20 000 × g for 30 minutes at 4 °C. The RNA pellet was dissolved with 30 μl of RNase-free water.
5
RNA Extraction from Granulosa and Theca Cells
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Total RNA was isolated from granulosa and theca samples after treatment with TRIzol LS Reagent (Invitrogen, Life Technologies Corporation), according to the manufacturer's instructions, with slight modifications. Also, adrenal samples were processed as positive controls. Briefly, 50-100 mg of tissue or pellet of granulosa cells was homogenized with 750 µL of TRIzol LS Reagent (Invitrogen) and incubated for 10 min at 25°C. Then, RNA was obtained by vigorously homogenizing with chloroform and incubating for 15 min at 4°C. After centrifugation at 12,000 g, the aqueous phase was incubated overnight with an equal volume of isopropyl alcohol at -20°C and centrifuged at 12,000 g to obtain the pellet of RNA, which was then washed with 75 vol/vol ethanol for 10 min at 4°C. Alcohol was replaced by DEPC water (Genbiotech) and prewarmed at 60°C. The extracted RNA was DNase treated with deoxyribonuclease I (Invitrogen) to eliminate contaminating DNA and was stored at -80°C for further use.
6
RNA Extraction from Saliva and Swabs
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For QIAmp MinElute Virus DNA/RNA Spin Kit (QVDRK) (Qiagen) and QIAmp Viral RNA Mini Kit (QVK)) (Qiagen) RNA extraction was performed following the manufacturer’s instructions. For Trizol LS Reagent (Thermo Fisher Scientific, #10296010) the manufacturer’s protocol was modified as follows: 1 ml of sample (i.e., saliva or nasal swab), 3 ml of Trizol LS Reagent and 0.6 ml of chloroform were mixed and centrifuged for 15 min at 12,000 × g at 4 °C. 0.8 ml of the colorless upper aqueous phase was carefully transferred to a new tube containing 1 ml isopropanol and 1 µl glycogen. The mixture was kept at − 20 °C overnight or was kept at room temperature for 10 min. After that, the mixture was span for 10 min at 12,000 × g, 4 °C, and the supernatant was discarded. The pellet was resuspended in 2 ml 75% ethanol, vortexed mixing and centrifuged for 5 min at 7500 g, 4 °C. The supernatant was discarded, and the pellet was incubated at room temperature for 5–10 min. After ethanol is evaporated, the pellet was resuspended in 50 µl RNase-free water, the quantity and quality of extraction was assessed using Qubit RNA HS kit, and stored at −80 °C.
7
Quantification of lncRNA AC129926.1 Expression
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Total serum RNA was extracted using TRIzol LS Reagent (Invitrogen, Carlsbad, CA). RNA was then reverse transcribed to generate cDNA utilizing an HiScript III RT SuperMix® for qPCR with gDNA Wiper (Vazyme, Nanjing, China). The expression level of AC129926.1 was detected by ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). With GAPDH as the internal reference, the expression of RNA was analyzed and quantified by the 2−ΔΔCt method. The primer sequences are shown in Table S1.
8
Quantifying Gene Expression in THP-1 Cells
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To quantify the expression of genes of interest, the THP-1 cells were grown in 24-well plates for 24 h with or without PQS/Pht/vitamin K addition. Total RNA was extracted using the TRIzol LS reagent (Invitrogen, California, USA). The qRT-PCR assay was performed using the ABI 7500 sequence detection system (Applied Biosystems, California, USA). Three replicates were performed with the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) as the internal control gene.
9
Quantifying miRNA and mRNA Expression
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TRIzol (Invitrogen, TRIzol™ LS Reagent, USA) extracted total RNA from serum, and after verifying the purity, the total RNA was reverse transcribed into cDNA according to the PrimeScriptTM RT reagent Kit (TaKaRa, Japan) instructions, for amplification reaction (TB Green® Fast qPCR Mix, TaKaRa, Japan). The design and construction of primer sequences were entrusted to Hunan Pulazete Biotech (as shown in Table 1 ). Reaction conditions (40 cycles) were 95°C, 5 min; 95°C, 15 s; 60°C, 34 s. 2-ΔΔCT calculated the relative expression of miR-18a-5p and ATM normalized against U6 and β-actin, respectively.
10
RNA Extraction and qRT-PCR Analysis
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RNA was extracted from the specimens by using Trizol LS reagent (Invitrogen, Carlsbad, CA) and RNeasy Mini Kit (Qiagen, Hilden, Germany) [52] , [53] (link), [54] (link). RT was carried out to generate cDNA by using an RT Kit (Applied Biosystems, Foster City, CA) as described in our published works [52] , [53] (link), [54] (link). PCR was performed to measure expressions of target genes by using a PCR kit (Applied Biosystems) on a Bio-Rad IQ5 Multicolor RT-PCR Detection System (Bio-Red, Hercules, CA). Expression levels of the genes were determined using comparative cycle threshold (CT) method with miR-1228 as an internal control. The targeted genes with CT values >35 were considered to be below the detection level of qRT-PCR [55] (link).
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