Gene expression profiling of whole blood: comparison of target preparation methods for accurate and reproducible microarray analysis.

Author(s): Vartanian K, Slottke R, Johnstone T, Casale A, Planck SR, Choi D, Smith JR, Rosenbaum JT, Harrington CA

Publication: BMC Genomics, 2009, Vol. 10, Page 2

PubMed ID: 19123946 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of storing blood frozen in PAXgene tubes, globin reduction, and template preparation method on the RNA transcriptome profiling. Specimens were stored at room temperature for 2 h or they were frozen at -80 degrees C for 1 week.

   Summary of Findings:

   The average RINs were 8.9 for fresh specimens and 8.8 for frozen specimens, but RIN were slightly reduced for fresh (8.6) and frozen (8.5) specimens after GLOBIN clear treatment. Treatment with GLOBIN clear or globin PNA oligos prior to one-cycle, or use of Ovation decreased the peaks associated with globin in the electropherogram compared to when specimens were prepared with one-cycle without globin depletion. Lower cRNA yields were observed after GLOBIN clear treatment than after the other treatments, but enough cRNA was generated using all methods for array hybridization. The percent present rate was 57.9% after globin PNA oligos with one-cycle, 56.1% in specimens treated with GLOBIN clear with one-cycle, 54.2% in specimens treated with Ovation, and 52.4% in specimens without globin reduction after one-cycle. One of the 6 specimens that used one-cycle without globin reduction was flagged for poor performance. After normalization, a high degree of correlation was observed in expression profiles between the different template preparation and globin reduction methods applied (r2>0.96). Use of globin PNAs with one-cycle resulted in the highest correlation of microarray and real-time PCR expression data (r2=0.77), followed by one-cycle without depletion (r2=0.75), ovation (r2=0.71) and GLOBIN clear with one cycle (r2=0.70). Generally a high degree of correlation was observed between expression profiles generated by microarray or real-time PCR for matched fresh and frozen specimens, but the correlations were slightly lower when specimens were prepared using globin PNAs. The highest reproducibility was observed using globin PNAs with one-cycle, but all methods had correlations between matched specimens of >0.97.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	DNA microarray
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	2 h 1 week
   DNA microarray Specific	Template modification	No modification GLOBIN clear Globin PNA oligos
   DNA microarray Specific	Nucleic acid amplification	Affymetrix one-cycle synthesis in vitro transcription Ovation amplification and labeling system

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