After the indicated treatments, experimental worms were harvested and washed with physiological saline at 4 °C and soaked immediately in QIAzol solution (catalog No. 79306, Qiagen, Hilden, Germany). Total RNA was extracted using the RNeasy Mini Kit (catalog No. 74104, Qiagen). Total RNA (200 ng) was treated with DNase and reverse-transcribed into cDNA using the iScript cDNA Synthesis Kit (catalog No. 12012801, Bio-Rad, Hercules, CA, USA), followed by the qRT-PCR analysis of CsGST expressions using gene-specific primers (Table S1 ) and the Rotor-Gene SYBR Green PCR Kit (catalog No. 204074, Qiagen) on a Rotor-Gene Q Real-time PCR System according to the manufacturer’s instructions. The qRT-PCR program included predenaturation for 5 min (94 °C), 40 cycles of amplification (94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s), and a melt cycle from 65 °C to 95 °C. Expression was evaluated in three independent biological samples with 3 technical repeats and normalized to C. sinensis tubulin expression (ΔCT). Fold induction (ΔΔCT) was calculated by comparison to non-stimulated controls. Data were analyzed with the Rotor-Gene Q ScreenClust HRM software using the 2−ΔΔCT method [53 (link)].
Qiazol solution
Manufactured by Qiagen
Sourced in Germany, United States
QIAzol solution is a reagent used for the isolation of total RNA from a variety of biological samples, including cells, tissues, and other sources. It is designed to effectively lyse samples and separate RNA from DNA, proteins, and other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy mini kit, by Qiagen (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Quantifast sybr green rt pcr kit, by Qiagen (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Rotor gene sybr green pcr kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Lmd7000, by Leica camera (1 mentions)
Nanodrop 1000, by Qiagen (1 mentions)
Rnaeasy plus micro kit, by Qiagen (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
Nsg mice, by Jackson ImmunoResearch (1 mentions)
Rnalater solution, by Qiagen (1 mentions)
Paxgene blood mirna kit, by Qiagen (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Qiagen (1 mentions)
19 protocols using qiazol solution
1
Quantifying CsGST Expression in Worms
2
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was isolated using QIAzol solution (Qiagen, Hilden, Germany), and then cDNA was synthesize with QuantiTect reverse transcription kit (Qiagen) according to the manufacturer’s guideline. For the real-time PCR, a total of 50 μL reaction mix constituted with 100 nM of cDNA template, 10 pM of each primers, and SYBR green master reagent (Go Taq, Promega, Madison, WI, USA) was prepared. Then, PCR was performed initial denaturation step at 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s by the TP 950 thermal cycler (Takara Bio, Shiga, Japan). All samples were analyzed twice. The results were normalized with GAPDH and then calculated by ΔΔCt method. Primer sequences for this experiment were provided in Supplementary Materials S2.
3
Harvesting and Lysing Cells for RNA Extraction
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All cell lines from each validated NB chemoresistance model were cultured and harvested accordingly. Following trypsin incubation step, each T25 flask was subjected to horizontal mechanical shock, necessary for dislodging all cells from the flask surface. The resultant cell suspension from each flask was transferred to an individually labelled, sterile 15 mL collection tube [BD Biosciences, USA] and centrifuged at 1500 rpm for five minutes to allow all cells to form a pellet at the base of the collection tube. The supernatant from each collection tube was aspirated by disposable, sterile glass pipette with due care to avoid inadvertent aspiration of cell pellet. Consequently, the remaining cell pellet was treated with 700 μL of Qiazol solution [Qiagen, Germany] in order to induce cell lysis. This step was performed within the confines of a fume cupboard and thorough pipette-mixing was applied for ensuring a resultant homogenous cell lysate suspension. Immediately after cell lysis induction, all cell lysate suspensions were snap frozen in liquid nitrogen solution and transferred to a − 80 °C freezer until RNA extraction was performed.
4
Isolation of Cholinergic Neurons for RNAseq
5
Hydrogel-Mediated Bone Regeneration in Mice
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Radial segmental defects in 8–10-week-old male NSG mice (Jackson Lab) were treated with 4.5% hydrogels functionalized with 1.0 mM adhesive peptide and crosslinked with 75:25 VPM:DTT with 15k hMSC (n = 7–8). The tissue within the 2.5 mm defect space was explanted at 1 week post-transplantation and stored in RNAlater solution (Qiagen) until further processing. Samples were placed in Qiazol solution (Qiagen), lysed by probe sonication, and homogenized in QIAshredder columns (Qiagen). Total RNA was isolated using an RNAeasy Plus Micro kit (Qiagen), and RNA content and purity were measured by spectrophotometry (NanoDrop 1000). cDNA synthesis was performed on total RNA (100 ng) using the High-Capacity RNA-to-cDNA Kit (ThermoFisher).
6
Exosomal RNA Isolation and Analysis
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RNA extracted from PAXgene tubes were processed using the PAXgene Blood miRNA Kit (Qiagen, Australia) according to manufacturers’ protocol. RNA from cell-free plasma and serum including exosome samples isolated via the UC method were extracted using the miRNeasy Mini Kit (Qiagen, Australia). The manufacturers’ protocol was followed with a slight modification of the protocol involving the use of Trizol LS (Life Technologies, Australia) instead of QIAzol solution (Qiagen, Australia). Briefly, the exosome pellet resuspended in 250 µl of PBS was lysed with 750 µl of Trizol LS and 200 µl of chloroform. After centrifugation, the aqueous phase was removed and the miRNeasy protocol was followed. In addition, a plasma/serum exosomal RNA isolation kit (Norgen Biotek (NG), Canada) was used to isolate exosomes and extract total exosomal RNA according to the manufacturers’ protocol. Exosomal RNA isolated via NG Kit are denoted as plasma NG and serum NG throughout the manuscript. RNaseA treatment (100 ng/ml, Qiagen, Australia) where indicated involved incubation of samples at 37°C for 10 minutes. The total RNA yield (comprising of mostly small RNA), composition and quality was analysed using the Agilent 2,100 Bioanalyser for small RNA profiles with the Small RNA kit (Agilent, Tokyo).
7
Endothelial Cell Response to LDL and Atorvastatin
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Human umbilical vein endothelial cells (Lonza, passages 3–5) were grown to 80% confluence in EGM-2 medium (Lonza) on type I collagen coated culture dishes. Where indicated, cells were incubated for 18 hours in human LDL cholesterol (100 μg/mL; Biomedical Technologies, Stoughton, MA, USA) and/or atorvastatin (100 nM; Sigma Scientific). TNFα (1 μg/mL; R&D Systems) was then added for an additional 18 hours after LDL cholesterol or atorvastatin treatment where indicated. Cells were then washed in warm PBS and lysed in QIAzol solution (Qiagen), and RNA was extracted following the manufacturer’s protocol. RNA quality was assessed by OD260/280 using a Nanodrop spectrophotometer. Total RNA was reverse transcribed using SuperScript III Reverse Transcriptase with oligo-dT primers (Invitrogen). Real-time PCR analysis was performed using SYBR green and Taq DNA polymerase (Qiagen) on a Bio-Rad CFX96 Connect Real-Time PCR Detection System. PCR primers for CX3CL1, GM-CSF, ICAM, VCAM, CX3CR1, and VEGF-A were obtained from Qiagen. RT-PCR data was normalized to GAPDH expression to identify relative changes in transcript levels.
8
Bulk RNA-seq Profiling of NR and HR Patients
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Methanol-fixed cells were equilibrated to 4°C, centrifuged for 5 minutes at 750g, rehydrated in Wash-Resuspension Buffer (0.04% BSA MilliporeSigma; 1mM DL-Dithiothreitol Solution, Invitrogen, Waltham, Massachusetts, USA; 0.2 U/μl Protector RNase Inhibitor, Thermo Fisher Scientific; 3x SSC Buffer, Naxo Ltd.), and lysed in 700µL QIAzol solution (Qiagen, Germantown, Maryland, USA). Methanol fixation and rehydration were performed according to the protocol approved by 10X Genomics (36 (link)). Bulk RNA was extracted from pooled cells of individual patients, with miRNeasy Micro kit (Qiagen), according to the manufacturer’s instructions. The quality and concentration of purified RNA were evaluated on 2100 Bioanalyzer with the RNA 6000 Pico kit (Agilent Technologies, Santa Clara, California, USA). Samples with RNA integrity number (RIN) ≥ 7 were considered eligible for further analysis. In total, the cells from 9 NR and 9 HR patients were used for further bulk RNA-seq.
9
Breast Milk Extracellular Vesicle RNA Isolation
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The breast milk extracellular vesicles were lysed in 500μl QIAzol solution (Qiagen) for 5 mins at room temperature, and RNA isolation was conducted using the Qaigen miRNeasy kit, as per the manufacturer’s instructions and without the optional Buffer RWT and second Buffer RPE washing steps. To ensure all RNA fragments were collected, the isolated RNA was eluted twice using 50μl RNase-free each time. The concentration of total RNA was measured using a NanoDrop 1000 instrument (Thermo Scientific, Wilmington, USA) and selected samples were analysed with Agilent Bioanalyzer 2100 using the Agilent RNA 6000 Nano Kit for total RNA and Small RNA kit for a focused review of the small RNA.
10
Automated Serum RNA Extraction
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Tubes were thawed for 2 hours at room temperature and then centrifuged for 10 minutes at 4500x g. The supernatant was removed immediately, and the pellets were resuspended in 4 mL RNase-free water and centrifugated again. 500 μL of QIAzol solution (Qiagen) was added to the obtained pellets. Total RNA, including small RNAs, was isolated from serum using a Qiagen miRNeasy mini kit on QIAcube for automated extraction following the protocol provided by the manufacturer (Qiagen GmbH D-40724 Hilden, Hoffmann-La Roche AG, Max-Volmer-Straße, Germany). The total elution volume was 50 μL of RNAse-free H2O.
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