title: Microarray analysis in clinical oncology: pre-clinical optimization using needle core biopsies from xenograft tumors.
abstract: BACKGROUND
  DNA microarray profiling performed on clinical tissue specimens can potentially provide significant information regarding human cancer biology. Biopsy cores, the typical source of human tumor tissue, however, generally provide very small amounts of RNA (0.3-15 microg). RNA amplification is a common method used to increase the amount of material available for hybridization experiments. Using human xenograft tissue, we sought to address the following three questions: 1) is amplified RNA representative of the original RNA profile? 2) what is the minimum amount of total RNA required to perform a representative amplification? 3) are the direct and indirect methods of labeling the hybridization probe equivalent?
  
  
  METHODS
  Total RNA was extracted from human xenograft tissue and amplified using a linear amplification process. RNA was labeled and hybridized, and the resulting images yielded data that was extracted into two categories using the mAdb system: "all genes" and "outliers". Scatter plots were generated for each slide and Pearson Coefficients of correlation were obtained.
  
  
  RESULTS
  Results show that the amplification of 5 microg of total RNA yields a Pearson Correlation Coefficient of 0.752 (N = 6,987 genes) between the amplified and total RNA samples. We subsequently determined that amplification of 0.5 microg of total RNA generated a similar Pearson Correlation Coefficient as compared to the corresponding original RNA sample. Similarly, sixty-nine percent of total RNA outliers were detected with 5 microg of amplified starting RNA, and 55% of outliers were detected with 0.5 microg of starting RNA. However, amplification of 0.05 microg of starting RNA resulted in a loss of fidelity (Pearson Coefficient 0.669 between amplified and original samples, 44% outlier concordance). In these studies the direct or indirect methods of probe labeling yielded similar results. Finally, we examined whether RNA obtained from needle core biopsies of human tumor xenografts, amplified and indirectly labeled, would generate representative array profiles compared to larger excisional biopsy material. In this analysis correlation coefficients were obtained ranging from 0.750-0.834 between U251 biopsy cores and excised tumors, and 0.812-0.846 between DU145 biopsy cores and excised tumors.
  
  
  CONCLUSIONS
  These data suggest that needle core biopsies can be used as reliable tissue samples for tumor microarray analysis after linear amplification and either indirect or direct labeling of the starting RNA.
authors.affiliation: Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, DHHS, Bethesda, MD, USA. goleye@mail.nih.gov
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Goley
authors.forename: Soni J
authors.initials: SJ
authors.lastname: Anderson
authors.forename: Cynthia
authors.initials: C
authors.lastname: Ménard
authors.forename: Eric
authors.initials: E
authors.lastname: Chuang
authors.forename: Xing
authors.initials: X
authors.lastname: Lü
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Tofilon
authors.forename: Kevin
authors.initials: K
authors.lastname: Camphausen
mesh_terms: D000818:Animals; D001707:Biopsy, Needle / Q000379:methods*; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012334:RNA, Neoplasm / Q000235:genetics*; D014183:Transplantation, Heterologous
chemical_list: D012334:RNA, Neoplasm