Humeral head articular cartilage gene expression was determined in 8 BtxA (4M, 4F), 8 Saline (4F, 4M), and 8 Control (4M, 4F) shoulders from 40-week old mice using established methods.32 Immediately following mouse sacrifice and dissection, tissues were frozen in liquid nitrogen and pulverized using a ball mill homogenizer (Mikro-Dismembrator U, Sartorius). RNA extraction was performed using guandinium thiocyanatephenol-chloroform (TRIzol, Thermo Fisher Scientific) and interphase separation (Phase Lock Gel, QuantaBio). RNA cleanup was performed using spin columns (RNeasy Mini Kit, Qiagen) with on-column deoxyribonuclease I treatment (Qiagen). RNA quantity and quality were determined using a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific). RNA was then reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Cartilage-, bone- and OA-related genes (Ihh, Acan, Runx2, Dkk1, Col2A1, Col10A1, BGLAP, ALPL, and BMP2) were evaluated by SYBR Green-based quantitative reverse transcription-polymerase chain reaction on an Applied Biosystems Quant-Studio 6 flex system. Glycer-aldehyde 3-phosphate dehydrogenase served as a housekeeping gene, and the relative mRNA expression level for each target gene was determined as 2-ΔΔCt.
Phase lock gel
Manufactured by Quantabio
Sourced in United States
Phase-lock gels are a laboratory product used to facilitate the separation and isolation of biomolecules, such as DNA, RNA, and proteins, during various experimental procedures. These gels are designed to create a physical barrier that helps to prevent the contamination or loss of the target analyte during specific steps of the extraction or purification process.
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Lab products found in correlation
Femto pulse system, by Agilent Technologies (7 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Qbit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Tissueruptor 2, by Qiagen (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Mikro dismembrator u, by Sartorius (1 mentions)
Deoxyribonuclease 1 treatment, by Qiagen (1 mentions)
Quantstudio 6 flex system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop 1000 spectrophotometer, by Agilent Technologies (1 mentions)
Nanodrop spectrophotometer, by Agilent Technologies (1 mentions)
12 protocols using phase lock gel
1
Murine Cartilage Gene Expression Analysis
2
Constructing a Genomic DNA Library
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The genomic DNA (gDNA) was isolated from 50 ml o/n culture of P putida KT2440 according to the protocol described in reference54 (link). Further purification was achieved using Phase Lock Gel (QuantaBio) with Phenol–Chloroform. After the centrifugation, isopropanol precipitation was repeated. The pellet was resuspended in water, the final amount was 60 µg DNA.
Plasmid DNA was purified as described in “Molecular biology methods ” from 12 ml E. coli DH5α cells. The plasmid DNA was diluted in water, the final amount was 10 µg DNA.
The library was constructed using SmaI (pBAD33 vector) and StuI (gDNA) for digestion resulting in an average size of 5 kb per insert (Bionexus, Inc.). After ligation, the plasmids were transformed into E. coli DH10 B (Lucigen). Quality control was done by restriction digest of library clones with BamHI. All restriction enzymes were produced by New England Biolabs, Frankfurt. The library was reisolated from E. coli DH10B as described in “Molecular biology methods ” and transferred into corresponding reporter strains.
Plasmid DNA was purified as described in “
The library was constructed using SmaI (pBAD33 vector) and StuI (gDNA) for digestion resulting in an average size of 5 kb per insert (Bionexus, Inc.). After ligation, the plasmids were transformed into E. coli DH10 B (Lucigen). Quality control was done by restriction digest of library clones with BamHI. All restriction enzymes were produced by New England Biolabs, Frankfurt. The library was reisolated from E. coli DH10B as described in “
3
Extraction of High-Quality RNA from Chordoma Tissue and Cell Cultures
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Snap-frozen chordoma tissue samples were pulverized while frozen, the tissue powder was lysed immediately with TRIzolate Reagent (Sigma-Aldrich) (50 mg sample per 500 uL reagent), and the samples were homogenized with a rotor-stator homogenizer. An equal volume of chloroform was added to the homogenates and vortexed, and the aqueous phase was separated using Phase Lock Gel™ (QuantaBio) (PLG 15 ml Heavy) tubes. RNA was isolated from the aqueous phase with the Direct-Zol RNA Miniprep kit (Zymo Research), according to the manufacturer's instructions, without DNase I treatment. RNA was prepared from the NP cell cultures with TRIzolate Reagent and Direct-Zol RNA Miniprep kit, according to the manufacturer's instructions. RNA quality was checked with Nanodrop 1000 spectrophotometer and Agilent 2100 Bioanalyzer – the data is summarized in Supplementary Table 2.
4
Isolation of High Molecular Weight Genomic DNA
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High molecular weight (HMW) genomic DNA (gDNA) was isolated from whole blood preserved in EDTA. 20 µL of whole blood was added to 2 ml of lysis buffer containing 10 mM Tris–HCl pH 8.0, 25 mM EDTA, 0.5% (w/v) SDS, and 100 µg/ml Proteinase K. Lysis was carried out at room temperature for a few hours until the solution was homogenous. The lysate was treated with 20 µg/ml RNase A at 37 °C for 30 min and cleaned with equal volumes of phenol/chloroform using phase lock gels (Quantabio, MA; Cat. # 2302830). DNA was precipitated by adding 0.4× volume of 5 M ammonium acetate and 3× volume of ice cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10 mM Tris, pH 8.0). Purity of gDNA was accessed using NanoDrop ND-1000 spectrophotometer and 260/280 ratio of 1.83 and 260/230 ratios of 2.34 was observed. DNA yield (120 µg total) was quantified using Qbit 2.0 Fluorometer (Thermo Fisher Scientific, MA). Integrity of the HMW gDNA was verified on a Femto pulse system (Agilent Technologies, CA).
5
High Molecular Weight Genomic DNA Extraction
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High molecular weight (HMW) genomic DNA (gDNA) extraction and nucleic acid library preparation were carried out by the University of California Davis DNA Technologies Core (Davis, CA). A whole-body sample from a male and a female B. lynchi was homogenized in 500 µl of homogenization buffer (10 mM Tris–HCl, pH 8.0 and 25 mM EDTA) using TissueRuptor II (Qiagen, Hilden, Germany; Cat. # 9002755). 500 µl of lysis buffer (10 mM Tris, 25 mM EDTA, 200 mM NaCl, and 1% SDS) and proteinase K (100 µg/ml) were added to the homogenate and incubated overnight at room temperature followed by RNAse A (20 µg/ml) treatment at 37 °C for 30 min. The lysate was cleaned with equal volumes of phenol/chloroform using phase-lock gels (Quantabio, Beverley, MA; Cat. # 2302830) and the DNA was precipitated by adding 0.4× volume of 5 M ammonium acetate and 3× volume of ice-cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10 mM Tris, pH 8.0). The purity of the DNA was accessed using NanoDrop spectrophotometer (260/280 and 260/230 ratios) and the integrity of the HMW gDNA was verified on a Femto pulse system (Agilent Technologies, Santa Clara, CA).
6
Hydrogel RNA Extraction and qPCR
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Hydrogels were removed from silicone molds using a pipette tip to scrape and transfer the gels into 1.5 mL Eppendorf tubes containing 500 µL of Trizol reagent (Invitrogen, Carlsbad, CA) on ice to extract RNA. The solution was then sonicated to allow complete break‐up of hydrogels for optimal RNA extraction. Phenol‐chloroform extraction was used to isolate RNA with Phase Lock Gels (Quantabio, Beverly, MA). A constant amount of RNA (0.1–1 µg) was reverse transcribed using the High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). 1 µg of cDNA in 5 µL of nuclease free water was then mixed with 10 µL of Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and run on the Applied Biosystems StepOnePlus Real Time PCR System. Primers used in this work are listed in Table S2 in the Supporting Information.
7
High Molecular Weight Genomic DNA Extraction
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High molecular weight (HMW) genomic DNA (gDNA) extraction and nucleic acid library preparation were carried out by the University of California Davis DNA Tech Core (Davis, CA). A whole-body sample from a female B. lindahli was homogenized in 500 µL of homogenization buffer (10 mM Tris–HCl pH 8.0 and 25 mM EDTA) using TissueRuptor II (Qiagen, Hilden, Germany; Cat. # 9002755). 500 µL of lysis buffer (10 mM Tris, 25 mM EDTA, 200 mM NaCl, and 1% SDS) and proteinase K (100 µg/mL) were added to the homogenate and incubated overnight at room temperature followed by RNAse A (20 µg/mL) treatment at 37 °C for 30 min. The lysate was cleaned with equal volumes of phenol/chloroform using phase-lock gels (Quantabio, Beverley, MA; Cat. #2302830) and the DNA was precipitated by adding 0.4× volume of 5 M ammonium acetate and 3× volume of ice-cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10 mM Tris, pH 8.0). The purity of the DNA was assessed using NanoDrop spectrophotometer (260/280 and 260/230 ratios) and the integrity of the HMW gDNA was verified on a Femto pulse system (Agilent Technologies, Santa Clara, CA).
8
High Molecular Weight Genomic DNA Isolation
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High molecular weight (HMW) genomic DNA (gDNA) was isolated from whole blood preserved in EDTA. Twenty microliter of whole blood was added to 2 ml of lysis buffer containing 100 mM NaCl, 10 mM Tris–HCl pH 8.0, 25 mM EDTA, 0.5% (w/v) SDS, and 100 µg/ml Proteinase K. Lysis was carried out at room temperature for a few hours until the solution was homogenous. The lysate was treated with 20 µg/ml RNase A at 37 °C for 30 min and cleaned with equal volumes of phenol/chloroform using phase lock gels (Quantabio, Massachusetts; Cat # 2302830). DNA was precipitated by adding 0.4× volume of 5 M ammonium acetate and 3× volume of ice-cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10 mM Tris, pH 8.0). Purity of gDNA was assessed using a NanoDrop ND-1000 spectrophotometer, and a 260/280 ratio of 1.83 and 260/230 of 2.35 were observed. DNA yield (120 µg total) was quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Massachusetts). Integrity of the HMW gDNA was verified on a Femto pulse system (Agilent Technologies, California), and 95% of the DNA was found in fragments larger than 120 kb in length.
9
High Molecular Weight Genomic DNA Extraction
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High molecular weight (HMW) genomic DNA (gDNA) extraction and nucleic acid library preparation were carried out by the University of California Davis DNA Technologies Core (Davis, CA). DNA was extracted from 50 mg of whole-body tissue using Nanobind tissue big DNA kit (Circulomics, Baltimore, MD; Cat. # SKU NB-900-701-01) following the manufacturer’s guidelines. Extracted DNA was cleaned with equal volumes of phenol/chloroform using phase-lock gels (Quantabio, Beverley, MA; Cat. #2302830) and precipitated by adding 0.4× volume of 5 M ammonium acetate and 3× volume of ice-cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10 mM Tris, pH 8.0). The purity of the DNA was accessed using NanoDrop spectrophotometer (260/280 and 260/230 ratios) and the integrity of the HMW gDNA was verified on a Femto pulse system (Agilent Technologies, Santa Clara, CA).
10
RNA Isolation and qPCR Analysis
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After washing the inside of the Transwell with 200 μl of PBS, the membrane was cut from the Transwell insert using a scalpel and placed in a 1.5 ml Eppendorf tube containing 500 μl of TRIzol reagent (Invitrogen, Carlsbad, CA) on ice to extract RNA. The solution was then vortexed to detach cells from membrane for optimal RNA extraction. Phenol-chloroform extraction was used to isolate RNA with Phase Lock Gels (Quantabio, Beverly, MA). A constant amount of RNA (0.1–1 μg) measured via NanoDrop (Thermo Fisher Scientific, Waltham, MA) was reverse transcribed using the high-capacity cDNA reverse transcription Kit (Applied Biosystems, Foster City, CA). For qPCR, 6.6 μl of cDNA in nuclease-free water was then mixed with 0.9 μl of a 5 μM forward and reverse primer pair (Integrated DNA Technologies, Coralville, IA) and 7.5 μl of Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and run on StepOnePlus Real Time PCR System (Applied Biosystems). CT values were calculated using the StepOnePlus software (v.2.3) and analyzed by the ΔCT method. Primers used for this work are listed in Table S2 of the supplementary material.
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