Chipping away at the chip bias: RNA degradation in microarray analysis.

Author(s): Auer H, Lyianarachchi S, Newsom D, Klisovic MI, Marcucci G, Kornacker K

Publication: Nat Genet, 2003, Vol. 35, Page 292-3

PubMed ID: 14647279 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to determine the effect of RNA degradation on transcript quantification by microarray analysis. RNA from peripheral blood mononuclear cells was degraded prior to processing by leaving the specimen at RT for 5 min or after processing by the addition of cellular extract. RNA degradation was determined using an author-developed model.

   Summary of Findings:

   Storing a specimen for 5 minutes at room temperature prior to RNA extraction or adding a cellular extract after RNA purification resulted in 75% of transcripts appearing to be differentially expressed by microarray analysis when compared to undegraded specimens. Importantly the false positives, but not the false negatives show a linear correlation with RNA degradation. The 3'/5' ratio of GAPDH and ACTB transcripts correlated strongly with degradation in aorta specimens (R2=0.65), but not well with bone marrow specimens (R2=0.16).

   Biospecimens

   • Bodily Fluid - Blood
   • Tissue - Skin
   • Tissue - Blood Vessel
   • Tissue - Bone Marrow
   • Cell - White Cells
   • Cell - Blood

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Peripheral blood mononuclear cells
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Snap frozen
   Analyte Extraction and Purification	Analyte isolation method	Trizol extraction immediately after thawing PBMC Trizol extraction after thawing and 5 min at room temperature Trizol extraction immediately following PBMC isolation Second trizol extraction following incubation of PBMC with endogenous RNAses
   Biospecimen Acquisition	Biospecimen location	Skin Natural killer cells Aorta Peripheral blood Bone marrow

   View more