Total RNA was collected and purified using RNAaqueous and DNase reagents according to the manufacturer’s instructions (Ambion, Houston, TX, USA). RNA quality was assessed using the Agilent 2100 BioAnalyzer, and samples with an RNA integrity number ≥ 6.0 were included for RNA sequencing. Up to 1 μg RNA was used to synthesize mRNA libraries using the TruSeq mRNA library kit (Illumina Inc, San Diego, CA, USA), as per the manufacturer’s instructions.
Rnaaqueous
Manufactured by Thermo Fisher Scientific
Sourced in United States
RNAaqueous is a RNA isolation kit designed to extract high-quality RNA from a variety of sample types. The kit utilizes a rapid, guanidinium-based lysis and purification method to efficiently isolate RNA while minimizing DNA and protein contamination.
Automatically generated - may contain errors
4 protocols using rnaaqueous
1
RNA Extraction and Sequencing
2
Total RNA Extraction and Sequencing
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Total RNA was collected at 7 days of treatment and purified using RNAaqueous and DNase reagents according to the manufacturer’s instructions (Ambion, Houston, TX, USA). RNA quality was assessed using the Agilent 2100 BioAnalyzer, and samples with an RNA integrity ≥6.0 were included for RNA sequencing. The synthesis of mRNA libraries was performed by Novogene Corporation Inc. according to their protocols. RNA library was formed by ployA capture (or rRNA removal), RNA fragmentation by covaris or enzyme digestion and reverse transcription of cDNA. Sequencing was performed as described below.
3
Total RNA Extraction and Sequencing
4
Quantitative Real-Time PCR Analysis
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Real-time PCR was performed as previously described [26 (link)]. Total RNA was collected and purified using RNAaqueous and DNase reagents according to manufacture’s protocol (Ambion, Houston, TX). cDNA prepared using Superscript reagents (BioRad, CA) and SYBR-green qPCR was performed using 5ng of cDNA, with primers designed using Beacon Designer software. Target gene expression was normalized using eukaryotic 18s rRNA as an endogenous control. All reactions were performed in triplicate and repeated three times.
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