RNA Extraction and Quantitative RT-PCR

As previously detailed [13 (link),14 (link),16 (link),17 (link),18 (link)], total RNA was extracted using the PureLink^TM RNA Mini kit (Life technologies, Carlsbad, CA, USA) and quantified by absorbance at A260/A280 nm (ratio >1.95) in a Nanodrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA). For quantitative RT-PCR experiments, 1.5 μg total RNA was reverse-transcribed with SuperScript VILO Master Mix (Life technologies). Messenger RNA levels of genes of interest were amplified, quantified, and normalized for the reference gene beta actin in a VIIA7 instrument (Life technologies).

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Tesi M., Bugliani M., Ferri G., Suleiman M., De Luca C., Bosi E., Masini M., De Tata V., Gysemans C., Cardarelli F., Cnop M., Eizirik D.L., Marchetti P, & Marselli L. (2021). Pro-Inflammatory Cytokines Induce Insulin and Glucagon Double Positive Human Islet Cells That Are Resistant to Apoptosis. Biomolecules, 11(2), 320.

Publication 2021

PureLinkTM RNA Mini kit Nanodrop 2000c spectrophotometer SuperScript VILO Master Mix VIIA7 instrument

Beta actin Gene Messenger rna Rt pcr

Corresponding Organization : University of Pisa

Other organizations : National Enterprise for NanoScience and NanoTechnology, Istituto Nanoscienze, KU Leuven, Université Libre de Bruxelles, Erasmus Hospital, Indiana Biosciences Research Institute

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Variable analysis

independent variables

Total RNA extraction method using the PureLink™ RNA Mini kit
Reverse transcription method using SuperScript VILO Master Mix

dependent variables

Messenger RNA levels of genes of interest

control variables

Quantification of total RNA by absorbance at A260/A280 nm (ratio >1.95) using Nanodrop 2000c spectrophotometer
Normalization of messenger RNA levels for the reference gene beta actin

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