The muscle samples from the P1 region were removed from −80°C and brought to a cryostat where they were allowed to come up to −20°C. The P1 region was chosen due to consistently presenting the most affected region of muscle in this RC injury model compared to the anterior and medial regions (Vargas-Vila et al., 2021 ). One notable difference is his region experiences the greatest fiber type changes with increased type II and decreased type I influencing the metabolic activity (Vargas-Vila et al., 2021 ). A 50–75 mg piece was removed from the center of each pinned region and placed in a pyrogen-free tube. RNA extraction was performed using the QIAGEN Fibrous Tissue mini kit on a QIAGEN Qiacube robot (QIAGEN, Germantown, MD, United States). In brief, the tissue was immersed in buffer RLT and disrupted by bead in the QIAGEN TissueLyser II (QIAGEN, Germantown, MD, United States), before being transferred to the Qiacube for RNA extraction. Samples were digested with Proteinase K, (QIAGEN, Germantown, MD, United States) prior to extraction. A DNase digestion step was included in the protocol. RNA was stored at −80°C.
Fibrous tissue mini kit
Manufactured by Qiagen
Sourced in United States, Germany
The Fibrous Tissue Mini Kit is a laboratory equipment product designed for the extraction and purification of nucleic acids (DNA/RNA) from fibrous tissue samples. The kit provides a standardized protocol and necessary reagents to efficiently isolate high-quality nucleic acids for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Tissuelyser 2, by Qiagen (2 mentions)
Proteinase k, by Qiagen (2 mentions)
Qiacube robot, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Rneasy mini, by Qiagen (2 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Brilliant ultra fast sybr qpcr master mix, by Agilent Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Tissuelyser lt, by Qiagen (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Stainless steel bead, by Qiagen (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Chloroform, by Merck Group (2 mentions)
Fs buffer, by Thermo Fisher Scientific (2 mentions)
Random primers, by Merck Group (2 mentions)
Qiacube, by Qiagen (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Human gapdh endogenous control, by Thermo Fisher Scientific (1 mentions)
15 protocols using fibrous tissue mini kit
1
Muscle Fiber Analysis in RC Injury
2
Rotator Cuff Injury Muscle RNA Extraction
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As previously described (Vasquez-Bolanos et al., 2021 (link)) the muscle samples from the P1 region were removed from −80°C and brought to a cryostat where they were allowed to come up to −20°C. The P1 region was chosen due to consistently presenting the most affected region of muscle in this rotator cuff injury model compared to the anterior and medial regions (Vargas-Vila et al., 2021 (link)). A 50–75 mg piece was removed from the center of each pinned region and placed in a pyrogen-free tube. RNA extraction was performed using the QIAGEN Fibrous Tissue mini kit on a QIAGEN Qiacube robot (QIAGEN, Germantown, MD). In brief, the tissue was immersed in buffer RLT and disrupted by bead in the QIAGEN TissueLyser II, (QIAGEN, Germantown, MD) before being transferred to the Qiacube for RNA extraction. Samples were digested with Proteinase K, (QIAGEN, Germantown, MD) prior to extraction. A DNase digestion step was included in the protocol. RNA was stored at −80°C.
3
Quantification of Cardiac Immune Cells
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RNA was isolated from mouse hearts using Qiagen’s Fibrous Tissue Mini Kit (Qiagen 74704) before concentration (Abs. 260) and quality (Abs. 260/280) of preps was assessed using a Nanodrop. cDNA was generated using the iScript cDNA synthesis kit (Biorad, #1708891). Quantitative real time PCR (qRT-PCR) was assessed with Taqman probes (CD45/Ptprc Mm01293577_m1; CD11b/Itgam Mm00434455_m1; F4/80/Adgre1 Mm00802529_m1; peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1α)/Ppargc1a Mm01208835_m1; nuclear respiratory factor 1 (NRF1)/Nrf1 Mm01135606_m1; estrogen-related receptor-a (ERRa)/Esrra Mm00433143_m1) and normalized against hypoxanthine phosphoribosyltransferase 1 (HPRT) (Hprt, Mm03024075_m1) to determine relative gene expression (RGE) using ΔΔCt as previously [77 (link), 78 ].
4
Luteolin Modulates Inflammatory Response
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Keratinocytes were pretreated with luteolin (10–100 µM, 30 min) before TNF stimulation (50 ng/mL, 6 h). Total RNA was extracted with an RNeasy Mini kit (Qiagen Inc., Valencia, CA). For human skin samples, total RNA was extracted using a Fibrous Tissue mini kit (Qiagen). An iScript cDNA synthesis kit (BioRad, Hercules, CA) was used for reverse-transcription of each sample. qRT-PCR was performed using Taqman gene expression assays (Applied Biosystems, Foster City, CA) for IL-6, IL-8, VEGF, NFKB1 and RELA. Samples were run using a 7300 Sequence Detector, according to TaqMan Gene Expression Assay instructions (Applied Biosystems). The mRNA expression was determined from standard curves run with each experiment. Relative mRNA levels were normalized to human GAPDH endogenous control (Applied Biosystems).
5
RNA Extraction and qPCR Analysis
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Total RNA was extracted by using RNeasy Mini or Fibrous Tissue Mini Kit (Qiagen) following manufacturer´s protocol. For cDNA synthesis, 1 or 3 μg of RNA was mixed with random hexamers and oligo-dT, 200 U M-MLV Reverse Transcriptase (Promega), 20 U RiboLock RNase inhibitor (Thermo Scientific), 0.5 mmol/L dNTP and reaction buffer (50 mmol/L Tris-HCl, 75 mmol/L KCl, 3 mmol/L MgCl2), incubated at +42°C for 1 hr, then at +70°C for 15 min and diluted 1:3 or 1:10 in sterile H2O to be used in qPCR (2 μl/reaction). Real-time qPCR was performed using Stratagene mx3005P (Agilent Technologies) or CFX96 (Bio-Rad) qPCR instruments and Brilliant Ultra-Fast SYBR QPCR Master mix (Agilent Technologies). DNA primer sequences are indicated in the Supplementary file 1 .
6
Isolation and Characterization of Total RNA from Fibrous Tissue
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Total RNA samples of left ventricles were isolated using a commercial isolation kit for fibrous tissues (Fibrous Tissue Mini Kit K74704, Qiagen). Briefly, mechanochemical tissue homogenization was performed in liquid nitrogen and several buffers using a tissue homogenizer system (Glas-Col, 099C-K5424). Following the proteinase incubation of the homogenate, total RNA from the samples were eluted in spin columns with several centrifuging and washing steps. Subsequently, the concentration and quality of total RNA samples were measured at 230 nm, 260 nm, and 280 nm (NanoDrop, ND-1000). The ratios of 260/280 and 260/230 were considered for the purity and quality of RNA and the extractions were repeated until the 1.8-2 ratio was achieved. All total RNA samples were run on 1% agarose gels to check their integrity. The observance of intact 28S and 18S RNA bands were required to continue further experiments with this sample (Fig. 2 ).
7
Lipodermatosclerosis Tissue Collection
8
RNA Isolation and qRT-PCR Analysis
9
RNA Extraction and cDNA Synthesis Protocol
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Tissue was quickly dissected and frozen on either dry ice or liquid nitrogen and stored at −80 °C. Tissue was homogenized in a Trizol reagent (QIAzol Lysis Reagent, Qiagen) using a stainless steel bead (Qiagen) and a TissueLyser LT (Qiagen) for 3 min at 20 Hz. Then, 200 μl chloroform (Sigma-Aldrich) was added and tubes were shaken vigorously for 15 sec and left at RT for 2 min, followed by centrifugation at 4 °C for 15 min at 12,000×g. The aqueous phase was mixed 1:1 with 70% ethanol and further processed using RNeasy Lipid Mini Kit following the instructions provided by the manufacturer. For muscle tissue, the lysis procedure, described the enclosed protocol in the Fibrous Tissue Mini Kit (Qiagen), was followed. After RNA extraction, RNA content was measured using a NanoDrop 2000 (Thermo Fisher) and 500 ng of RNA was converted into cDNA by mixing FS buffer and DTT (Thermo Fisher) with Random Primers (Sigma-Aldrich) and incubated for 3 min at 70 °C followed by addition of dNTPs, RNase out, Superscript III (Thermo Fisher) and placed in a thermal cycler for 5 min at 25 °C, 60 min at 50 °C, 15 min at 70 °C, and kept at −20 °C until further processing.
10
Total RNA Extraction and cDNA Synthesis for qPCR
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Total RNA was extracted using an RNeasy Mini or Fibrous Tissue Mini kit (Qiagen) following the manufacturer´s protocols. For cDNA synthesis, 1 to 3 μg of RNA was mixed with random hexamers and oligo-dT, 200 U M-MLV Reverse Transcriptase (Promega), 20 U RiboLock RNase inhibitor (Thermo Scientific), 0.5 mM dNTP and reaction buffer (50 mM Tris–HCl, 75 mM KCl, 3 mM MgCl2), incubated at +42 °C for 1 h, then at +70 °C for 15 min and diluted 1:3 to 1:10 in sterile H2O and used in qPCR (2 μl/reaction). Real-time qPCR was performed using a Stratagene mx3005P qPCR instrument (Agilent Technologies) and Brilliant Ultra-Fast SYBR QPCR Master mix (Agilent Technologies). DNA primer sequences are indicated in the Supplemental Table I.
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