title: Variability of protein level and phosphorylation status caused by biopsy protocol design in human skeletal muscle analyses.
abstract: BACKGROUND
  Bergström needle biopsy is widely used to sample skeletal muscle in order to study cell signaling directly in human tissue. Consequences of the biopsy protocol design on muscle protein quantity and quality remain unclear. The aim of the present study was to assess the impact of different events surrounding biopsy protocol on the stability of the Western blot signal of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), Akt, glycogen synthase kinase-3β (GSK-3β), muscle RING finger protein 1 (MuRF1) and p70 S6 kinase (p70 S6K). Six healthy subjects underwent four biopsies of the vastus lateralis, distributed into two distinct visits spaced by 48 hrs. At visit 1, a basal biopsy in the right leg was performed in the morning (R1) followed by a second in the left leg in the afternoon (AF). At visit 2, a second basal biopsy (R2) was collected from the right leg. Low intensity mobilization (3 × 20 right leg extensions) was performed and a final biopsy (Mob) was collected using the same incision site as R2.
  
  
  RESULTS
  Akt and p70 S6K phosphorylation levels were increased by 83% when AF biopsy was compared to R1. Mob condition induced important phosphorylation of p70 S6K when compared to R2. Comparison of R1 and R2 biopsies revealed a relative stability of the signal for both total and phosphorylated proteins.
  
  
  CONCLUSIONS
  This study highlights the importance to standardize muscle biopsy protocols in order to minimize the method-induced variation when analyzing Western blot signals.
authors.affiliation: Centre de recherche, Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Québec, Canada. Richard.debigare@rea.ulaval.ca.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Caron
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Charette
authors.forename: François
authors.initials: F
authors.lastname: Maltais
authors.forename: Richard
authors.initials: R
authors.lastname: Debigaré
references.citation: Oncogene. 2005 Nov 14;24(50):7401-9
references.pmid: 16288287
references.citation: Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):7987-92
references.pmid: 9653127
references.citation: Nat Cell Biol. 2003 Feb;5(2):87-90
references.pmid: 12563267
references.citation: Am J Respir Crit Care Med. 2001 Nov 1;164(9):1712-7
references.pmid: 11719314
references.citation: Physiol Rev. 2009 Apr;89(2):381-410
references.pmid: 19342610
references.citation: Physiology (Bethesda). 2008 Jun;23:160-70
references.pmid: 18556469
references.citation: Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):38-43
references.pmid: 10618367
references.citation: Am J Respir Crit Care Med. 2007 Aug 1;176(3):261-9
references.pmid: 17478621
references.citation: J Nutr. 1999 Jan;129(1S Suppl):227S-237S
references.pmid: 9915905
references.citation: Expert Opin Emerg Drugs. 2009 Dec;14(4):619-32
references.pmid: 19860537
references.citation: Am J Respir Crit Care Med. 2002 Sep 15;166(6):809-13
references.pmid: 12231489
references.citation: Med Sci Sports Exerc. 1998 Oct;30(10):1467-74
references.pmid: 9789845
references.citation: Eur J Appl Physiol. 2008 Sep;104(1):57-65
references.pmid: 18535836
references.citation: J Physiol. 2006 Nov 1;576(Pt 3):923-33
references.pmid: 16916907
references.citation: Am J Physiol Endocrinol Metab. 2008 Sep;295(3):E595-604
references.pmid: 18577697
references.citation: FASEB J. 2004 Jan;18(1):39-51
references.pmid: 14718385
references.citation: Endocrinology. 2005 Jun;146(6):2726-35
references.pmid: 15746249
references.citation: Lancet. 1997 Apr 12;349(9058):1050-3
references.pmid: 9107242
references.citation: Am J Physiol Endocrinol Metab. 2008 Feb;294(2):E241-50
references.pmid: 17971516
references.citation: Toxicol Sci. 2005 May;85(1):647-56
references.pmid: 15673845
references.citation: Science. 2001 Nov 23;294(5547):1704-8
references.pmid: 11679633
references.citation: J Physiol. 2007 Jul 15;582(Pt 2):813-23
references.pmid: 17478528
references.citation: J Cell Biol. 2009 Jun 15;185(6):1083-95
references.pmid: 19506036
references.citation: Biochem Biophys Res Commun. 2010 Oct 8;401(1):104-11
references.pmid: 20833131
references.citation: J Immunol Methods. 2010 Feb 28;353(1-2):148-50
references.pmid: 20035759
references.citation: J Physiol. 2008 Nov 15;586(22):5589-600
references.pmid: 18818241
references.citation: J Mol Biol. 2008 Mar 7;376(5):1224-36
references.pmid: 18222470