Systematic Identification of the Optimal Housekeeping Genes for Accurate Transcriptomic and Proteomic Profiling of Tissues following Complex Traumatic Injury

Methods and Protocols

article

Andrea H. Dragon¹  Cassie J. Rowe¹  Alisha M. Rhodes¹  Olivia L. Pak¹  Thomas A. Davis¹  Elsa Ronzier¹ 

[1] Cell Biology and Regenerative Medicine Program, Department of Surgery, Uniformed Services University of the Health Sciences;Henry M. Jackson Foundation for the Advancement of Military Medicine

关键词: trauma;    gene expression;    housekeeping gene (HKG);    reference gene normalization;    gene expression stability;    immunoblot;    real-time quantitative polymerase chain reaction (RT-qPCR);    secondary organs;    rat;   

DOI  :  10.3390/mps6020022

学科分类：电力

来源: mdpi

【 摘 要 】

Trauma triggers critical molecular and cellular signaling cascades that drive biological outcomes and recovery. Variations in the gene expression of common endogenous reference housekeeping genes (HKGs) used in data normalization differ between tissue types and pathological states. Systematically, we investigated the gene stability of nine HKGs (Actb, B2m, Gapdh, Hprt1, Pgk1, Rplp0, Rplp2, Tbp, and Tfrc) from tissues prone to remote organ dysfunction (lung, liver, kidney, and muscle) following extremity trauma. Computational algorithms (geNorm, Normfinder, ΔCt, BestKeeper, RefFinder) were applied to estimate the expression stability of each HKG or combinations of them, within and between tissues, under both steady-state and systemic inflammatory conditions. Rplp2 was ranked as the most suitable in the healthy and injured lung, kidney, and skeletal muscle, whereas Rplp2 and either Hprt1 or Pgk1 were the most suitable in the healthy and injured liver, respectively. However, the geometric mean of the three most stable genes was deemed the most stable internal reference control. Actb and Tbp were the least stable in normal tissues, whereas Gapdh and Tbp were the least stable across all tissues post-trauma. Ct values correlated poorly with the translation from mRNA to protein. Our results provide a valuable resource for the accurate normalization of gene expression in trauma-related experiments.

【 授权许可】

CC BY