TY - JOUR
T1 - Systematic Identification of the Optimal Housekeeping Genes for Accurate Transcriptomic and Proteomic Profiling of Tissues following Complex Traumatic Injury
AU - Dragon, Andrea H.
AU - Rowe, Cassie J.
AU - Rhodes, Alisha M.
AU - Pak, Olivia L.
AU - Davis, Thomas A.
AU - Ronzier, Elsa
N1 - Funding Information:
This work was supported by the Department of Defense Congressionally Directed Medical Research Program (CDMRP) under the Peer-Reviewed Orthopaedic Research Program (PRORP; Grant Award #: W81XWH1920032).
Publisher Copyright:
© 2023 by the authors.
PY - 2023/2/23
Y1 - 2023/2/23
N2 - 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.
AB - 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.
KW - gene expression
KW - gene expression stability
KW - housekeeping gene (HKG)
KW - immunoblot
KW - rat
KW - real-time quantitative polymerase chain reaction (RT-qPCR)
KW - reference gene normalization
KW - secondary organs
KW - trauma
UR - http://www.scopus.com/inward/record.url?scp=85153782048&partnerID=8YFLogxK
U2 - https://doi.org/10.3390/mps6020022
DO - https://doi.org/10.3390/mps6020022
M3 - Article
C2 - 36961042
AN - SCOPUS:85153782048
SN - 2409-9279
VL - 6
JO - Methods and Protocols
JF - Methods and Protocols
IS - 2
M1 - 22
ER -