Total RNA (including sRNA) was extracted from the serum samples using the miRNeasy Serum/Plasma kit (Qiagen) according to manufacturer's protocol. This kit is specifically designed for the purification of cell-free RNA from serum and plasma. The quality and concentration of the resulting RNA samples were determined using a Nanodrop Spectrophotometer (NanoPhotometer N60, Implen). In addition, a more detailed analysis of the sRNAs isolated from the samples used for sequencing was performed using the Bioanalyzer Small RNA kit (Agilent Technologies, Inc.).
Bioanalyzer small rna kit
Manufactured by Agilent Technologies
Sourced in United States
The Bioanalyzer small RNA kit is a lab equipment product from Agilent Technologies. It is designed for the analysis and quantification of small RNA molecules, such as microRNA, siRNA, and other non-coding RNA species, in a sample. The kit provides a sensitive and automated platform for assessing the size, quantity, and quality of small RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Spin x centrifuge tubes, by Merck Group (2 mentions)
Next seq 50 bp single end reads, by Illumina (2 mentions)
Mini protean tbe urea gel, by Bio-Rad (2 mentions)
Mirneasy serum plasma kit, by Qiagen (1 mentions)
Hitrap q hp anion exchange column, by Cytiva (1 mentions)
Truseq small rna library kit, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
5 protocols using bioanalyzer small rna kit
1
Serum sRNA Extraction and Analysis
2
Plasma miRNA Isolation and Quantification
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For clinical samples, miRNA was isolated from 600 µl of plasma with Macherey-Nagel’s NucleopSpin® miRNA Plasma kit (740981.250) following the manufacturer’s protocol. Plasma miRNAs were eluted in 30 μL nuclease-free water. The concentration and the quality of the extracted miRNAs were quantified using ThermoFisher’s Qubit® microRNA Assay Kit (Q32880) and analyzed using Agilent’s BioAnalyzer® Small RNA Kit (5067-1548), respectively. For miRQC experiments, plasma miRNAs from three healthy donors were pooled together for further experiments. Synthetic hsa-let-7a-5p, hsa-let-7b-5p and hsa-let-7c-5p miRNAs (IDT) were spiked into MS2-phage RNA without endogenous let-7 miRNAs (Sigma-Aldrich, #Roche-10165948001) to a final concentration of 5 × 106 copies/µg RNA (for samples 9–11).
3
Small RNA Sequencing from Drosophila Ovaries
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For small RNA sequencing, two replicates per strain were prepared. Small RNA was isolated from 50 pairs of ovaries using HiTrap Q HP anion exchange columns (Cytiva, Velizy-Villacoublay, France) as described in [37 (link)], and the eluate was run on a 10% TBE urea gel (Thermo Fisher Scientific). Small RNA size selection (18–50 bp) was performed on gel at the sequencing facility. Quality was checked with the Bioanalyzer small RNA kit (Agilent, Santa Clara, CA, USA). Library construction was performed using the TruSeq Small RNA Library kit (Illumina, San Diego, CA, USA) and sequenced (1 × 50 single reads) on an Illumina HiSeq 4000 at the IGBMC Microarray and Sequencing facility. Adapter sequences were removed using cutadatp [38 (link)]. Size selection was then performed using PRINSEQ lite version 0.20.4 [39 (link)]. All subsequent analyses were built upon small RNA counts after normalization according to the miRNA amounts, as described in [34 (link)].
4
Desiccation Stress-Induced Small RNA Profiling
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Total RNA was isolated using Trizol (Thermo Fisher Scientific) from three replicates of 30 4–7 day-old female flies after desiccation stress and under control conditions. RNA samples were treated with DNAse I (Thermo Fisher Scientific) following manufacturer’s instructions. Small RNAs were obtained by gel size selection from total RNA using 15% Mini-Protean TBE-Urea Gel (Bio-Rad, #4,566,056). The gel was run at 300 V for 50 min. Fragments between 17 and 30 nucleotides were carefully cut from the gel and were put in an Eppendorf with 250 μL NaCl 0.5 M and then were placed at 4 °C in a rotating wheel o/n. Next, the sample was transferred to Corning Costar Spin-X centrifuge tubes (Merck, CLS8162), spinned and cleaned with standard ethanol washes, and eluted in DEPEC-H2O. Quality check was performed using Bioanalyzer small RNA kit (Agilent). Libraries were sequenced using Illumina Next-Seq 50 bp single-end reads.
5
Desiccation Stress-Induced Small RNA Profiling
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Total RNA was isolated using Trizol (Thermo Fisher Scientific) from three replicates of 30 4–7 day-old female flies after desiccation stress and under control conditions. RNA samples were treated with DNAse I (Thermo Fisher Scientific) following manufacturer’s instructions. Small RNAs were obtained by gel size selection from total RNA using 15% Mini-Protean TBE-Urea Gel (Bio-Rad, #4,566,056). The gel was run at 300 V for 50 min. Fragments between 17 and 30 nucleotides were carefully cut from the gel and were put in an Eppendorf with 250 μL NaCl 0.5 M and then were placed at 4 °C in a rotating wheel o/n. Next, the sample was transferred to Corning Costar Spin-X centrifuge tubes (Merck, CLS8162), spinned and cleaned with standard ethanol washes, and eluted in DEPEC-H2O. Quality check was performed using Bioanalyzer small RNA kit (Agilent). Libraries were sequenced using Illumina Next-Seq 50 bp single-end reads.
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