After 10 DIV, each culture was washed by gently replacing the growth medium with PBS warmed to 37 °C. Each culture well was then evaluated visually for GFP expression levels. Wells showing infection levels <70% were discarded. The remaining wells were subjected to two further 37 °C PBS washes. After the third wash, PBS was aspirated away, and the bottom part of the insert (axonal domain) was scraped using a sterile swab tip (TX761MD, Tex-wipe). The scraping tip of the swab was then cut and placed into Qiazol reagent (Qiagen) for 1–2 min, before the Qiazol reagent was transferred into a new collection tube. The inner part of the membrane (somatic domain) was repeatedly washed with 0.7 ml of Qiazol and placed into a collection tube.
Qiazol reagent
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, Netherlands, France, Spain, Italy, Canada, Japan, China
QIAzol reagent is a proprietary solution developed by Qiagen for the isolation of high-quality RNA from a variety of biological samples. It is designed to effectively lyse cells and tissues while maintaining the integrity of the extracted RNA.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (53 mentions)
Mirneasy mini kit, by Qiagen (41 mentions)
Rneasy mini kit, by Qiagen (32 mentions)
Nanodrop, by Thermo Fisher Scientific (24 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (22 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (20 mentions)
Lightcycler 480, by Roche (19 mentions)
Quantitect reverse transcription kit, by Qiagen (18 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (17 mentions)
Rneasy lipid tissue mini kit, by Qiagen (15 mentions)
Rneasy kit, by Qiagen (15 mentions)
Iscript cdna synthesis kit, by Bio-Rad (14 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (14 mentions)
Sybr green master mix, by Thermo Fisher Scientific (13 mentions)
Iq sybr green supermix, by Bio-Rad (12 mentions)
Miscript 2 rt kit, by Qiagen (12 mentions)
2100 bioanalyzer, by Agilent Technologies (12 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (12 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (12 mentions)
Mirneasy kit, by Qiagen (11 mentions)
556 protocols using qiazol reagent
1
Harvesting Axon and Soma RNA Samples
2
Isolation and Analysis of Small RNAs
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Small RNAs were isolated while using the miRNeasy Mini Kit (QIAGEN, Toronto, ON, Canada) following the Quick-Start protocol of miRNeasy Mini Kit manufacturer’s instructions. Briefly, tracheal rings from TOC samples (stored in RNAlater) were collected, lysed in 700 µL of QIAzol reagent (QIAGEN, Toronto, ON, Canada), and homogenized for two minutes using 0.5 mm glass beads (Biospec Products Inc., Bartlesville, OK, USA) and a tissue homogenizer (MP FastPrep-24 Classic Instrument, MP Biomedicals, Solon, OH, USA). The isolated EVs from EV samples were likewise lysed in 700 µL of QIAzol reagent (QIAGEN, Toronto, ON, Canada). The samples were then deproteinized in chloroform and centrifuged for 15 min. at 12,000× g at 4 °C. The upper aqueous portion of the samples was precipitated in 95% ethanol. The samples were sequentially washed and centrifuged with RWT and RPE buffers using RNeasy Mini columns in 2 mL collection tubes (QIAGEN, Toronto, ON, Canada). Finally, the purified RNA was eluted in 27 μL RNase-free water and quality control of RNA was performed using the RNA ScreenTape Analysis kit (Agilent Technologies, Santa Clara, CA, USA) and the Agilent 4200 TapeStation Analysis Software A.02.01 SR1 (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer’s instruction. The samples were then stored at −80 °C.
3
Serum miRNA Expression Analysis
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Total RNA was isolated from the serum of patients or cells using the QIAzol™ reagent (Qiagen, Inc.) as previously described (18 (link),19 (link)). The miRNA expression analysis was performed using qPCR analysis as previously reported (18 (link),19 (link)). Briefly, serum samples were mixed at a ratio of 1:10 with QIAzol™lysis reagent and vortexed for 1 min using a mini vortexer (Thermo Fisher Scientific, Inc). Cell pellets (~106 cells) were mixed with 1 ml QIAzol™ reagent (Qiagen, Inc.). The lysates were extracted using CHCl3 and the aqueous phase was further processed, removing phenol and other contaminants, to obtain total RNA enriched in miRNA using the miRNAeasy Mini Kit (Qiagen, Inc.). miRNA levels were determined following conversion of RNA to cDNA using the RT2 miRNA first strand kit (Qiagen, Inc.) followed by amplification of cDNA in the Applied Biosystems 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.) using SYBR qPCR reaction mixture and miRNA specific primers (Qiagen, Inc.). Amplification of cDNA was performed under the following conditions: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec (18 (link),19 (link)). U6 spliceosomal RNA served as an internal control, and data was quantified using the comparative Cq method (20 (link)).
4
Quantitative RT-PCR Analysis of Cartilage Gene Expression
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Total RNA from articular cartilage and chondrocyte cultures was isolated using QIAzol reagent (Qiagene). One μg total RNA was subjected to reversed transcription. Amplification was performed using a ABI 7900 Detection System (Applied Biosystems), with 2 × TaqMan® Universal PCR Master Mix. Primers for Atg4, Atg12, p62, Beclin, collagen II, aggrecan, SOX9, IL-1β, CXCL9, and calibrator gene 18S rRNA (Supplementary Table 1 ). Changes in mRNA expressions were calculated as 2−ΔΔCt, where ΔΔCt = ΔCtACLT – ΔCtsham and ΔCt = Ctgene − Ct18S.
5
RNA Expression Analysis in Macrophages
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Total RNA in macrophage cultures and bone tissue was isolated using QIAzol reagent (Qiagene, Valencia, CA, USA). Upon reverse transcription of 1 μg total RNA, mRNA expression was detected using primers for NFATc1 (forward: 5′-GAAGGTGTACTCCTCGGGTGG-3′; reverse: 5′-GATACCTGGCTCGGTAACACCAC-3′), V-ATPase (forward: 5′-AGAAAGCCAAGTGCCTACTCC-3′; reverse: 5′-AAAGGGAAGGGTTTCTTTTGG-3′), MMP9 (forward: 5′-GGGAAGGCTCTGCTGTTCA-3′; reverse: 5′-CGGTTGAAGCAAAGAAGGAG-3′), cathepsin K (forward: 5′-CCTGCGGCATTACCAACAT-3′; reverse: 5′-GCTGCAGGACTCCAATGTCT-3′), TNFSF13b (forward: 5′-TTCCATGGCTTCTCAGCTTT-3′; reverse: 5′-CGTCCCCAAAGACGTGACT-3′), and actin (forward: 5′-GACGGCCAGGTCATCACTAT-3′; reverse: 5′-CTTCTGCATCCTGTCAGCAA-3′). Equation 2−ΔΔCt, where ΔΔCt = ΔΔCtglucocorticoid − ΔCtvehicle and ΔΔCt = Ctgene − Ctactin was adopted to calculated the relative expression of each gene.
6
RNA, DNA, and Protein Extraction from Brain Tissue
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Brain tissue was homogenized and lysed in 1 mL QIAzol reagent (Qiagen) using a TissueRuptor homogenizer (Qiagen). RNA was then chloroform extracted and purified using the RNeasy lipid tissue mini kit (Qiagen). The quality of the RNA was verified by spectrometry and visualization of ribosomal RNA bands on an agarose gel. DNA was precipitated from the remaining interphase and organic phase with 75% ethanol, and the protein in the supernatant was then isopropanol precipitated, denatured with 0.3M guanidine hydrochloride, and resuspended in 1% SDS.
7
mRNA Extraction and Quantification from mDCs
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After 20 hr of cell culture, mDCs were lysed with Qiazol reagent (Qiagen, Hilden, Germany) and homogenized with QIAshredder columns before storage at −80° pending extraction of mRNA using an miRNeasy Mini Kit (Qiagen) according to an adapted manufacturer's protocol with an off‐column DNA digest with TurboDNase (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) and RNeasy MinElute Cleanup Kit (Qiagen). Messenger RNA was quantified using a NanoDrop ND‐1000 Spectrophotometer (ThermoScientific, Thermo Fisher Scientific, Waltham, MA, USA) and converted to cDNA using RevertAid Reverse Transcriptase and complementary reagents (Fermentas, ThermoScientific). Relative quantification of target genes relative to the 18S housekeeping gene was conducted in triplicates by real‐time quantitative PCR using Taqman Universal PCR MasterMix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and an Applied Biosystems Viia 7 real‐time thermal cycler. Results were analysed using viia 7 software (Applied Biosystems). Taqman primers were purchased from Applied Biosystems.
8
Molecular Characterization of Bone, Cartilage, and Brain
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The previous method was used with modification [17] (link), [20] (link). Prior to homogenization, femur and tibia were harvested and medullary fluid was washed out. Articular cartilage from distal femur and proximal tibia was pooled for total RNA extraction. For brain, tissues were dissected into three regions including FC, VMH, and BS. The tissues containing dorsal, medial and caudal raphe were labeled as BS (Fig. 1E ). The mRNA expressions of each gene were determined using quantitative real-time PCR with the primers listed in Table 1 . Expression of collagen types I and II was evaluated for synthesis of bone and cartilage matrix, while NGFß as a neurotrophic factor. As a marker for serotonergic signaling, expression of tph2 was assayed with Sim1 and REST as its potential regulators and Pet 1, Lhx8, and RGS as its downstream effectors [21] (link), [22] (link). Total RNA was extracted using an RNeasy Lipid Tissue mini kit with QIAzol reagent (Qiagen) and chloroform (Fisher Scientific). Reverse transcription was performed, and real-time PCR was carried out using ABI 7500 with SYBR green PCR kits (Applied Biosystems). The mRNA level of GAPDH was used as an internal control.
9
Quantitative Analysis of miRNA Expression
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Total RNA was extracted from cell pellets or tissues using QIAzol reagent (Qiagen, Hilden, Germany). Semi-quantitative RT-PCR was performed using Hot-start DNA polymerase (Invitrogen) with the housekeeping gene β-actin as an internal control. Quantitative real-time PCR was performed using SYBR Green master mixture on HT7900 system (Applied Biosystems, Foster City, CA). The expression level of mature miR-29b was quantified by TaqMan microRNA assays (Applied Biosystems). The comparative ΔCt method was used to calculate the relative abundance of miRNA compared with RNU6B expression (Fold difference relative to RNU6B).
10
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was prepared using the Qiazol reagent (Qiagen, Valencia, CA, USA), and 2 μg of the isolated RNA was reverse transcribed into cDNA using Superscript II Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, lnc., Waltham, MA, USA). Quantitative real-time PCR analysis was performed in triplicate with a Rotor-Gene Q (Qiagen) with SYBR Green (Qiagen). Expression levels were normalized against endogenous glyceraldehyde 3-phosphate dehydrogenase levels. The following primers were used: Gadph, 5′-TGA CCA CAG TCC ATG CCA TCA CTG-3′ and 5′-CAG GAG ACA ACC TGG TCC TCA GTG-3′; cfos, 5′-ATG GGC TCT CCT GTC AAC ACA-3′ and 5′-TGG CAA TCT CAG TCT GCA ACG CAG-3′; Nfatc1, 5′-CTC GAA AGA CAG CAC TGG AGC AT-3′ and 5′-CGG CTG CCT TCC GTC TCA TAG-3′; Acp5, 5′-CTG GAG TGC ACG ATG CCA GCG ACA-3′ and 5′-TCC GTG CTC GGC GAT GGA CCA GA-3′; Runx2, 5′-CCC AGC CAC CTT TAC CTA CA-3′ and 5′-CAG CGT CAA CAC CAT TC-3′; Alpl, 5′-CAA GGA TAT CGA CGT GAT CAT G-3′ and 5′-GTC AGT CAG GTT CCG ATT C-3′; Ibsp, 5′-GGA AGA GGA GAC TTC AAA CGA AG-3′ and 5′-CAT CCA CTT CTG CTT CGT TC-3′; Bglap, 5′-ATG AGG ACC CTC TCT CTG CTC AC-3′ and 5′-AGA GCA AAC TGC AGA AGC TGA GAG-3′; and Amh, 5′-GGA GTC TGC AGC ACT GAC TC-3 and 5′-TCA CTT CAG CCA GAT GTA GG-3′.
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