RNA Extraction and Quantitative PCR

Total RNA was extracted using Geneaid Minikit (Geneaid). After digestion with DNase, 1 μg of RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega) and random hexamers (Promega) and used as template for conventional PCR analyses using GoTaq enzyme (Promega), before analysis on agarose or acrylamide gels and quantification by densitometry using Image Lab Software (Biorad).⁵⁵ (link) Real-time quantitative PCRs (qPCR) were performed using the SYBR Green I Master and the LightCycler 480 System (Roche). All primers used are listed in the Table S7.

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Ruta V., Naro C., Pieraccioli M., Leccese A., Archibugi L., Cesari E., Panzeri V., Allgöwer C., Arcidiacono P.G., Falconi M., Carbone C., Tortora G., Borrelli F., Attili F., Spada C., Quero G., Alfieri S., Doglioni C., Kleger A., Capurso G, & Sette C. (2024). An alternative splicing signature defines the basal-like phenotype and predicts worse clinical outcome in pancreatic cancer. Cell Reports Medicine, 5(2), 101411.

Publication 2024

M-MLV reverse transcriptase random hexamers GoTaq enzyme Image Lab Software SYBR Green I Master LightCycler 480 System

Corresponding Organization : Agostino Gemelli University Polyclinic

Other organizations : Vita-Salute San Raffaele University, University Hospital Ulm

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Variable analysis

independent variables

RNA extraction using Geneaid Minikit
DNase digestion
Reverse transcription using M-MLV reverse transcriptase and random hexamers
Conventional PCR using GoTaq enzyme
Real-time quantitative PCR (qPCR) using SYBR Green I Master and the LightCycler 480 System

dependent variables

Gene expression levels measured on agarose or acrylamide gels and quantified by densitometry using Image Lab Software (Biorad)

control variables

Not explicitly mentioned

controls

Positive control: Not specified
Negative control: Not specified

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