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
A
B
Divide powder into aliquots
G
RNA analysis,
4 plaques
Plaque
powder
A
RNA extraction
Protein analysis,
5 plaques
Plaque
powder
B
C
Tissue extraction
E
D
~10 mm
Manual pulverization
of tissue
A
F
Protein and lipid
extraction
Lipid analysis,
10 plaques
Plaque
powder
B
Isolation of RNA
followed by analysis
and comparison
between aliquots
Isolation of protein
followed by analysis
and comparison
between aliquots
see Figure 3A
see Figure 3B
A
B
Isolation of lipids followed
by analysis and
comparison between
aliquots
see Figure 3C