Total tissue RNA was isolated using the DNA/RNA Mini kit (Qiagen, Hilden, Germany), as described by Alanazi et al [70 (link)]. Briefly, tissues were lysed in 350 μllysis buffer with 3.5 μl β-mercaptoethanol. The lysate was subsequently filtered and homogenized with 70% ethanol, and the DNA was loaded onto the column and digested with DNase at room temperature for 30 minutes. Afterwards, the column membrane was washed, and the RNA was eluted with 40μl RNase-free H2O and stored in nuclease-free collection tubes at -80°C. The concentration, purity, and quality of the isolated RNA were all determined using the Agilent 2100 Bio analyzer system and Agilent Small RNA analysis kit according to the instructions provided by the manufacturer (Agilent Technologies, Waldbronn, Germany).
Agilent small rna analysis kit
Manufactured by Agilent Technologies
Sourced in Germany
The Agilent Small RNA analysis kit is a tool for the analysis of small RNA molecules, such as microRNA, siRNA, and piRNA. The kit provides reagents and protocols for the isolation, detection, and quantification of small RNA species from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Agilent bioanalyzer 2100 system, by Agilent Technologies (4 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Dna rna mini kit, by Qiagen (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Mirneasy ffpe kit, by Qiagen (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nucleospin mirna plasma kit, by Macherey-Nagel (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
7500 fast real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Cultured cell total rna purification mini kit, by Favorgen Biotech (1 mentions)
6 protocols using agilent small rna analysis kit
1
RNA Isolation and Quality Assessment
2
Extraction and Quantification of miRNAs from FFPE Tissue and Serum
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Formalin-fixed, paraffin-embedded biopsy samples were deparaffinized and underwent total RNA and miRNA extraction using miRNeasy FFPE Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. miRNA yield was determined through a NanoDrop ND-1000 spectrophotometer (CELBIO), and the quality assessed by agarose gel electrophoresis.
Total miRNAs were isolated from thawed serum samples using NucleoSpin miRNA Plasma kit (Macherey-Nagel, Düren, Germany). Total miRNAs were extracted from 300 μL, 600 μL or 900 μL of serum and further eluted in 30 μL of nuclease-free water. The miRNA concentration and quality were assessed with the Bioanalyzer 2100 (Agilent Technologies, CA) using the Agilent Small RNA Analysis kit (Agilent, CA).
Total miRNAs were isolated from thawed serum samples using NucleoSpin miRNA Plasma kit (Macherey-Nagel, Düren, Germany). Total miRNAs were extracted from 300 μL, 600 μL or 900 μL of serum and further eluted in 30 μL of nuclease-free water. The miRNA concentration and quality were assessed with the Bioanalyzer 2100 (Agilent Technologies, CA) using the Agilent Small RNA Analysis kit (Agilent, CA).
3
FFPE lncRNA Extraction and Analysis
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FFPE tissue samples were deparaffinized and underwent total RNA and lncRNA extraction using miRNeasy FFPE Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. LncRNA yield was determined through a Qubit™ 3.0 Fluorometer (Thermo Fisher Scientific), and the quality was assessed by agarose gel electrophoresis. The lncRNA concentration and quality were assessed with the Bioanalyzer 2100 (Agilent Technologies, CA) using the Agilent Small RNA Analysis kit (Agilent, CA).
4
Extraction and Characterization of Total RNA from Colon Tissues
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Total RNA was extracted from 40 colon cancer tissues and 40 matched normal colon tissues using an AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. The isolated RNA concentration, purity, and quality were determined using an Agilent 2100 Bioanalyzer system and Agilent Small RNA analysis kit (Agilent Technologies, Waldbronn, Germany) according to the manufacturer’s instructions.
5
Quantifying Colon Cancer Cell TLR2 Expression
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As described previously,30 (link)–32 (link) total RNA was extracted from colon cancer cells or from colon tissue using a DNA/RNA Mini kit from Qiagen NV (Venlo, The Netherlands) according to the manufacturer’s instructions. To quantify the RNA concentration and verify RNA purity and quality, we used the Agilent 2100 Bioanalyzer system and Agilent Small RNA analysis kit according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA) and a Nano-Drop8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthetized from 1 µg of each RNA sample with a high-capacity cDNA reverse transcription kit from Applied Biosystems (Thermo Fisher Scientific). The real-time PCR (qPCR) was performed as previously described30 (link) using SYBR Green Master Mix from Bio-Rad Laboratories Inc. (Hercules, CA, USA) following the manufacturer’s instructions. The number of copies of the TLR2 gene was measured using the Applied Biosystems 7,500 Fast real-time PCR detection system. GAPDH served as the internal control. The primers used for real-time PCR are presented in Table S1 . Each reaction was conducted in triplicate. The specificity of each primer pair was verified by the presence of a single melting temperature peak, and the results were analyzed using the 2−∆∆Ct (Livak) relative expression method.
6
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted using the Cultured Cell Total RNA Purification Mini Kit (Favorgen, Germany). After extraction, total RNA concentration and purity were evaluated using an Agilent 2100 Bio-analyzer system and an Agilent Small RNA analysis kit (Agilent technologies, Waldbronn, Germany). RNA (1 μg per sample) was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit from Applied Biosystems (Warrington, USA) at 37 °C for 2 h.
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