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Molecular Analysis in Mechanobiology
Published in Jiro Nagatomi, Eno Essien Ebong, Mechanobiology Handbook, 2018
The final step before the actual performance of RT-PCR is to verify the RNA quality and measure its concentration. RNA degradation can be a source of large experimental error in RT-PCR. One method of RNA qualification is the assessment of ribosomal RNA integrity by denaturing agarose gel electrophoresis. rRNA accounts for approximately 75%–80% of total RNA and the 28S and 18S rRNAs are easily detected by ethidium bromide staining. Intact preparations are characterized by sharp 28S and 18S rRNA bands and an approximately 2:1 ratio in staining intensity. RNA degradation is recognized by a decrease in this ratio and “smearing,” staining of a broad range of lower molecular weight fragments. More recently, electrophoresis has been miniaturized and accelerated using lab-on-chip technologies such as the Agilent Bioanalyzer. The Bioanalyzer measures 28S:18S ratios, as well as calculating an RNA integrity number (RIN) ranging from 1 to 10 that is an objective measurement derived from the entire electrophoresis profile [39,40]. While lab-on-chip technology is quicker, the costs associated with the instrument and consumables are considerably greater.
Transcriptome analysis of Takifugu obscurus liver in response to acute retene exposure
Published in Journal of Environmental Science and Health, Part A, 2020
Shulun Jiang, Di-an Fang, Dongpo Xu
Total RNA was extracted using Trizol reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. Quality of RNA was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). RNA samples with a 28 S/18 S ratio higher than 1.0 and RNA integrity number (RIN) higher than 7.2 were considered qualified.