RNA was isolated from 77 placenta samples using Qiagen RNeasy Kit (Qiagen). After homogenization of the placental tissue sample, the RNA extraction was performed following the manufacturer’s protocol. Only 160 µL of the watery phase was combined with an equal amount of 70% ethanol and loaded onto the RNeasy Mini Column. The RNA was eluted with 40 µL RNase-free water. The RNA concentration, size range and quality were measured using Agilent Bioanalyser 2100, Eukaryote Total RNA Nano Series II according to the manufacturer’s protocol (Agilent RNA 6000 Nano Kit Guide). A Qubit Fluorometric Quantitation Assay was used to validate RNA concentration. Samples used for RNA sequencing data analysis had a mean RIN of 5.1 (±1.09 SD).
Agilent rna 6000 nano kit guide
Manufactured by Agilent Technologies
Sourced in United States
The Agilent RNA 6000 Nano Kit Guide is a laboratory product that provides a standardized procedure for the analysis of RNA samples using the Agilent 2100 Bioanalyzer system. The kit includes reagents, chips, and a guide for performing RNA analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy kit, by Qiagen (1 mentions)
2100 bioanalyser, by Agilent Technologies (1 mentions)
Loading dye, by New England Biolabs (1 mentions)
Flash gel rna cassette system, by Lonza (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000 spectrophotometer v3, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Bio-Rad (1 mentions)
Nanodrop 2000 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
5 protocols using agilent rna 6000 nano kit guide
1
Placenta RNA Isolation and Characterization
2
Comprehensive RNA Characterization Protocol
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DNA samples and 1 kilobases plus DNA marker were mixed with 6 × loading dye (New England BioLabs, USA), loaded onto a 1% agarose gel in Tris–acetate-EDTA buffer and run at 110 V for 50 min. RNA samples were run and analysed using the FlashGel RNA cassette system (Lonza Bioscience, USA).
Additionally, saRNA samples were prepared according to the Agilent RNA 6000 Nano Kit guide and run on the Agilent Bioanalyzer 2100 using the mRNA Nano assay class protocol. The data was analysed by the 2100 Expert software (B.02.10.S1764). Fraction of intact saRNA based on the electrogram was represented by area under the curve (Supplementary information). The Lonza RNA marker (Lonza Bioscience, USA, #50,577) was used.
Additionally, saRNA samples were prepared according to the Agilent RNA 6000 Nano Kit guide and run on the Agilent Bioanalyzer 2100 using the mRNA Nano assay class protocol. The data was analysed by the 2100 Expert software (B.02.10.S1764). Fraction of intact saRNA based on the electrogram was represented by area under the curve (Supplementary information). The Lonza RNA marker (Lonza Bioscience, USA, #50,577) was used.
3
RNA Extraction and cDNA Synthesis
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Total RNA was purified as previously described in [23 ]. RNA samples were quantified using NanoDrop 1000 Spectrophotometer V3.8 (Thermo Fisher Scientific), and their quality was checked using an Agilent RNA 6000 Nano Kit Guide (Agilent Technologies, Santa Clara, U.S.A.). VILO cDNA Synthesis Kit (Invitrogen, Monza and Brianza, Italy, #11754050) was used to convert RNA into cDNA. Then, 50 ng of cDNA was added with 1X SYBR Green PCR Master Mix (Bio-Rad #1708880), according to the manufacturer's instructions. Primers used are listed in Supplementary File Table 4 .
4
RNA Extraction from Tissue Sections
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Twenty micrometer tissue sections were scraped from glass slides using a sterile scalpel. Total RNA was isolated using the Trizol reagent and protocol. RNA samples were analyzed using protocols and supplies described in Agilent RNA 6000 Nano Kit guide (Cat. #5067-1511). RNA integrity (RIN) values were generated with the Agilent 2100 bioanalyzer (software ver. B.02.07.SI532; Agilent Technologies, Santa Clara, CA, USA).
5
RNA Sample Characterization Protocol
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The RNA samples isolated were run and evaluated in a 1% agarose gel. A260 and A280 values were measured using a nano‐spectrophotometer (NanoDrop® 2000/2000c Spectrophotometer; Thermo Fisher Scientific®). Consequently, they were analyzed by automated electrophoresis (2100 Bioanalyzer®; Agilent®) and their RNA integrity number (RIN) values were calculated. For the nano‐spectrophotometric and the automated electrophoretic evaluations, the protocols defined by the manufacturer's were followed strictly (Thermo Fisher Scientific® NanoDrop® 2000/2000c Spectrophotometer V1.0 User Manual; Agilent® RNA 6000 Nano® Kit Guide). The agarose gel images of randomly selected 20 RNA samples are shown in Supporting Information: 2 Figure. Concentration values, A260/A280 ratios, and RIN values of the purified RNA samples are presented in Supporting Information: 3 File.
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