1. A B C D E F G RNA analysis, 4 plaques Protein analysis, 5 plaques
Supplemental Figure 1. Manual grinding of atherosclerotic plaque preserved in RNAlater® into fine powder (GEMS). Sample was maintained at all times submerged in RNAlater® solution until processed. Before grinding a ceramic mortar and pestle were pre-chilled on dry-ice (A) and a sample was allowed to freeze in the vessel (B). Sample was first carefully crushed into smaller pieces (C) before grinding into a finer powder (D). For RNA isolation the powder was overlaid with TRIzol® solution (E). For protein isolation the powder was transferred into a tube containing extraction buffer (F). If required the frozen powder can be preserved for later analysis. For a detailed protocol see Materials and Methods section. (G) A general scheme of the experimental design for evaluation of compositional equivalence of sample aliquots.
Intact Tissue
Manual pulverization
of tissue
Isolation of RNA followed by analysis and comparison between aliquots
~10 mm
Tissue extraction A B C D E F
RNA extraction
Protein and lipid
extraction G
Divide powder into aliquots
Isolation of protein followed by analysis and comparison between aliquots
Isolation of lipids followed by analysis and comparison between aliquots
RNA analysis,
4 plaques
Protein analysis,
5 plaques
Lipid analysis,
10 plaques
Plaque
powder
see Figure 3A
see Figure 3B
see Figure 3C