RNA degradation and contamination was monitored on 1% agarose gels. According to the manufacturer's protocol, each tissue sample was washed three times using cold PBS and 1 ml TRIzol® reagent was added (Thermo Fisher Scientific, Inc.) to extract the RNA. The RNA concentration was measured using the Qubit® RNA assay kit in a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.). RNA integrity was verified using an RNA Nano 1000 assay kit for the Bioanalyzer 2100 system (Agilent Technologies, Inc.). The method for determining the levels of lncRNAs and miRNAs was the same as that used for mRNAs. For the quantification of circRNAs, exonuclease was used to exclude non-circRNAs. The RNA was divided into two copies. Linear RNA was digested with RNase R (cat. no. RNR07250; Epicentre; Illumina, Inc.) to leave only the circRNAs. The other half of the sample from the same RNA extraction was not treated with RNase R. The two samples of RNA were reverse transcribed according to a previous study (25 (link)). The sample treated with RNase R was used to examine the expression of circRNAs and the other sample that was not treated with RNase R was used to measure the expression of β-actin.
Rna nano 1000 assay kit
Manufactured by Agilent Technologies
Sourced in United States
The RNA Nano 1000 assay kit is a tool used for the analysis and quantification of RNA samples. It provides reliable and accurate measurements of RNA concentration and quality within the range of 25-1000 ng/µL. The kit utilizes microfluidic technology to analyze small sample volumes, making it suitable for research applications that require efficient use of limited samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Ribo zero rrna removal kit, by Illumina (1 mentions)
2100 series, by Agilent Technologies (1 mentions)
2 protocols using rna nano 1000 assay kit
1
RNA Extraction and Circular RNA Analysis
2
RNA Extraction and Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) was employed to isolate and purify total RNA according to the manufacturer’s instructions. The amount and purity of total RNA in each sample was analyzed using a bioanalyzer (2100 series; Agilent Technologies, Santa Clara, CA, USA) and an RNA Nano1000 Assay Kit (Agilent Technologies) so that the RNA integrity number (RIN) > 7.0. Ribosomal RNA was removed according to the instructions of the Ribo-Zero™ rRNA Removal Kit (Illumina). At 95 °C, the remaining RNA was broken into short fragments using divalent cations. Subsequently, cDNA was created by reverse transcription using fragmented RNA as a template, followed by addition of deoxynucleoside triphosphate, RNase H, and Escherichia coli DNA polymerase I to synthesize the second strand. After purification of AMPure XP beads (Beckman Coulton, Fullerton, CA, USA) and end repair, poly(A) was added, and sequencing connectors were attached. Then, cDNA of a certain length range was extracted. PCR amplification was performed to obtain a cDNA library. The average insert size of the final cDNA library was 300 ± 50 bp. Twelve separate RNA-seq libraries were generated for the study group and control group.
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