Gene and microRNA Expression Analysis

RNA was extracted from tissues and cells using Trizol Reagent (Thermo Fisher), miRNeasy or RNeasy Mini Kit (Qiagen), and mRNA levels of gene expression were quantified by qPCR using SYBR Green (GeneCopoeia) on a 7500 Real-Time PCR machine (Applied Biosystems). mRNA levels were normalized to ribosomal protein, large, P0 (36B4), and the relative fold change compared to the controls was calculated. Primers were designed and synthesized by IDT (Integrated DNA Technologies); sequences are listed in Supplementary Table 1. MicroRNAs were extracted from cells and tissues using miRNeasy Mini Kit (Qiagen). Reverse transcription was done with TaqMan MicroRNA Reverse Transcription Kit and quantified using a TaqMan MicroRNA assay kit and TaqMan Universal Master Mix II, no UNG (Thermo Fisher) for real-time-PCR, then normalized to small nuclear RNA U6 (U6), with fold change calculated and compared to the control as described [74 (link)].

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Juguilon C., Wang Z., Wang Y., Enrick M., Jamaiyar A., Xu Y., Gadd J., Chen C.L., Pu A., Kolz C., Ohanyan V., Chen Y.R., Hardwick J., Zhang Y., Chilian W.M, & Yin L. (2022). Mechanism of the Switch from NO to H2O2 in Endothelium-Dependent Vasodilation in Diabetes. Basic research in cardiology, 117(1), 2.

Publication 2022

Trizol Reagent miRNeasy or RNeasy Mini Kit SYBR Green 7500 Real-Time PCR machine miRNeasy Mini Kit TaqMan MicroRNA Reverse Transcription Kit TaqMan MicroRNA assay kit TaqMan Universal Master Mix II

Assay Cells Gene expression Microrna Mrna Primers Real time pcr Reverse transcription Ribosomal protein Sybr green Tissues Trizol U6 small nuclear rna

Corresponding Organization :

Other organizations : Northeast Ohio Medical University

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Variable analysis

independent variables

RNA extraction method (Trizol Reagent, miRNeasy, or RNeasy Mini Kit)

dependent variables

MRNA levels of gene expression
MicroRNA levels

control variables

Ribosomal protein, large, P0 (36B4) for mRNA normalization
Small nuclear RNA U6 for microRNA normalization

controls

Positive control: Not specified
Negative control: Not specified

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