Tissues were collected at appropriate time points and snap-frozen in liquid nitrogen. Total RNA was isolated using a Qiagen RNeasy Plus Isolation Kit followed by cDNA synthesis (High Capacity cDNA RT kit, Applied Biosystems, 4374966). RT-PCR was performed using TaqMan Universal Master Mix II (Fisher, 4440038), according to manufacturer’s protocol. Primers used were: Myc (Fisher, Mm00487804_m1) and Tbp (Fisher, Mm00446973_m1). Samples were analysed in triplicate on an Eppendorf Mastercycler Realpex 2 with with Eppendorf Quantstudio design & Statistical Analysis Vl.2 Software. Tbp was used as an internal amplification control.
Rneasy plus isolation kit
Manufactured by Thermo Fisher Scientific
The RNeasy Plus Isolation Kit is a product designed for the purification of high-quality total RNA from a variety of sample types. It utilizes a silica-membrane-based technology to capture and purify RNA molecules, enabling efficient recovery of RNA for downstream applications.
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2 protocols using rneasy plus isolation kit
1
Quantitative RT-PCR Analysis of Myc Expression
2
Pancreatic Cytokine Profiling
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Pancreata were collected at appropriate time points and snap-frozen in liquid nitrogen. Whole pancreas protein extracts were isolated and incubated with mouse inflammation/cytokine antibody arrays (40 targets, Abcam, ab133999; 97 targets, Abcam ab169820), according to manufacturer's instructions. Signal intensity was determined using Image J software. Differential expression of cytokine RNA was assessed by quantitative real time-PCR (RT-PCR). For this, total pancreas RNA was isolated using a Qiagen RNeasy Plus Isolation Kit followed by cDNA synthesis (High Capacity cDNA RT kit, Applied Biosystems, 4374966). RT-PCR was performed using TaqMan Universal Master Mix II (Fisher, 4440038), according to manufacturer's protocol.
Primers used were: Cxcl5 (Fisher, Mm00441260_m1), Gas6 (Fisher Mm00441242_m1), Ccl9 (Fisher, Mm00436451_g1), Ccl2 (Fisher, Mm00490378_m1), Tbp (Fisher, Mm00446973_m1), Shh (Fisher, Mm00436528_m1). Samples were analyzed in triplicate on an Eppendorf Mastercycler Realpex 2 with accompanying software. Tbp was used as an internal amplification control.
Primers used were: Cxcl5 (Fisher, Mm00441260_m1), Gas6 (Fisher Mm00441242_m1), Ccl9 (Fisher, Mm00436451_g1), Ccl2 (Fisher, Mm00490378_m1), Tbp (Fisher, Mm00446973_m1), Shh (Fisher, Mm00436528_m1). Samples were analyzed in triplicate on an Eppendorf Mastercycler Realpex 2 with accompanying software. Tbp was used as an internal amplification control.
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