title: High-throughput simultaneous analysis of RNA, protein, and lipid biomarkers in heterogeneous tissue samples.
abstract: BACKGROUND
  With expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze, from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD).
  
  
  METHODS
  We used a SilverHawk atherectomy catheter to collect atherosclerotic plaques from patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater®, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured.
  
  
  RESULTS
  The quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r = 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting.
  
  
  CONCLUSIONS
  The TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.
authors.affiliation: Merck Sharp & Dohme Corporation, Whitehouse Station, NJ, USA. Vladimir.Reiser@ge.com
authors.forename: Vladimír
authors.initials: V
authors.lastname: Reiser
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Smith
authors.forename: Jiyan
authors.initials: J
authors.lastname: Xue
authors.forename: Marc M
authors.initials: MM
authors.lastname: Kurtz
authors.forename: Rong
authors.initials: R
authors.lastname: Liu
authors.forename: Cheryl
authors.initials: C
authors.lastname: Legrand
authors.forename: Xuanmin
authors.initials: X
authors.lastname: He
authors.forename: Xiang
authors.initials: X
authors.lastname: Yu
authors.forename: Peggy
authors.initials: P
authors.lastname: Wong
authors.forename: John S
authors.initials: JS
authors.lastname: Hinchcliffe
authors.forename: Michael R
authors.initials: MR
authors.lastname: Tanen
authors.forename: Gloria
authors.initials: G
authors.lastname: Lazar
authors.forename: Renata
authors.initials: R
authors.lastname: Zieba
authors.forename: Marina
authors.initials: M
authors.lastname: Ichetovkin
authors.forename: Zhu
authors.initials: Z
authors.lastname: Chen
authors.forename: Edward A
authors.initials: EA
authors.lastname: O'Neill
authors.forename: Wesley K
authors.initials: WK
authors.lastname: Tanaka
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Marton
authors.forename: Jason
authors.initials: J
authors.lastname: Liao
authors.forename: Mark
authors.initials: M
authors.lastname: Morris
authors.forename: Eric
authors.initials: E
authors.lastname: Hailman
authors.forename: George Y
authors.initials: GY
authors.lastname: Tokiwa
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Plump
mesh_terms: D015415:Biomarkers / Q000032:analysis; D015925:Cryopreservation; D004210:Dissection; D006801:Humans; D008055:Lipids / Q000032:analysis* / Q000302:isolation & purification; D058226:Plaque, Atherosclerotic / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D013048:Specimen Handling / Q000379:methods*; D014020:Tissue Extracts / Q000737:chemistry; D014021:Tissue Preservation / Q000379:methods*
chemical_list: D015415:Biomarkers; D008055:Lipids; D011506:Proteins; D012333:RNA, Messenger; D014020:Tissue Extracts; D012313:RNA