Blood and bronchoalveolar fluid specimens were collected in RNA later RNA Stabilization Reagent (Qiagen GmbH) tubes and then stored at −80°C until further analysis. Total RNA, including miRNAs, was extracted from BALFs and whole blood using TRIZOL reagent (Invitrogen; Thermo Fisher Scientific, Inc.) as described in the manufacturer's protocol. To evaluate the concentration and purity of RNA, we used NanoDrop w ND‐1000. The RNA integrity number (RIN) was assessed using the Agilent 2100 Bioanalyzer with RNA 6000 series II Nano Lab Chip analysis kit (Agilent Technologies, Inc.). The 2100 Bioanalyzer generates numerical RIN values. RIN is an incremental scale spanning 0–10, with 10 indicating maximum RNA integrity. All RNA samples extracted from BALFs and blood samples were of high integrity as judged by the obtained RIN values of higher than eight. Then, all samples with a ratio (28S/18S) of above 1.8, OD 260/280 ratio greater than 1.9, and RIN >8 were sent to BGI company in China for sequencing. In this regard, small RNA libraries were generated from the purified RNA using the Illumina Small RNA v1.5 sample preparation kit (Illumina, Inc.), according to the manufacturer's instructions.
Rna 6000 series 2 nano labchip analysis kit
Manufactured by Agilent Technologies
Sourced in Germany
The RNA 6000 series II Nano LabChip analysis kit is a laboratory equipment product that enables the analysis of RNA samples. It provides quantitative and qualitative assessment of RNA samples, including measurement of concentration and integrity.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nanodrop 1000 spectrophotometer, by Avantor (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Rnalater rna stabilization reagent, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Small rna v1.5 sample preparation kit, by Illumina (1 mentions)
3 protocols using rna 6000 series 2 nano labchip analysis kit
1
Extraction and Evaluation of High-Quality RNA
2
Assessing RNA Quality from Tissue and Plasma
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The concentration of the isolated RNA from tissue specimens was assessed by measuring the optical density at 260 nm (OD260) and 280 nm (OD280) using a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) and a Nano-Drop 1000 spectrophotometer (PEQLAB Biotechnologie GmbH), whereas the quality of the RNA purified from plasma could only be assessed by PCR amplification curves and efficiencies, due to the absence of ribosomal RNA. The purity of RNA was evaluated by the ratio of the absorbance at OD260 to OD280. Analysis of the RNA integrity number (RIN) was conducted by use of the Agilent 2100 Bioanalyzer with RNA 6000 series II Nano LabChip analysis kit (both from Agilent Technologies, Inc.). The 2100 Bioanalyzer generates numerical RIN values. RIN is an incremental scale which spans from 0 to 10, whereas increasing RNA integrity correlates with increasing RIN value. All total RNA samples extracted from CRC and adjacent neoplasm-free mucosal tissues were of high integrity as judged by the obtained RIN values of ≥8.0.
3
RNA Purity and Integrity Assessment for CRC
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The concentration of the isolated RNA from tissue specimens was assessed by measuring the optical density at 260 nm (OD260) and 280 nm (OD280) using a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a NanoDrop 1000 spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany), whereas the quality of the RNA purified from plasma was assessed by PCR amplification curves and efficiencies, due to the absence of ribosomal RNA. RNA purity was evaluated by the ratio of the absorbance at OD260/OD280. Analysis of RNA integrity (RIN) was conducted using the Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Waldbronn, Germany) with the RNA 6000 series II Nano LabChip analysis kit. The 2100 Bioanalyzer generates numerical RIN values. RIN is an incremental scale spanning 0–10, where increasing RIN correlates with increasing RIN value. Total RNA samples extracted from CRC and adjacent neoplasm-free mucosal tissues were of high integrity as judged by the obtained RIN values of ≥8.0.
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