Characteristics of cross-hybridization and cross-alignment of expression in pseudo-xenograft samples by RNA-Seq and microarrays

Camilo Valdes, Pearl Seo, Nicholas Tsinoremas, Jennifer Clarke

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Background: Exploring stromal changes associated with tumor growth and development is a growing area of oncologic research. In order to study molecular changes in the stroma it is recommended to separate tumor tissue from stromal tissue. This is relevant to xenograft models where tumors can be small and difficult to separate from host tissue. We introduce a novel definition of cross-alignment/cross-hybridization to compare qualitatively the ability of high-throughput mRNA sequencing, RNA-Seq, and microarrays to detect tumor and stromal expression from mixed 'pseudo-xenograft' samples vis-à-vis genes and pathways in cross-alignment (RNA-Seq) and cross-hybridization (microarrays). Samples consisted of normal mouse lung and human breast cancer cells; these were combined in fixed proportions to create a titration series of 25% steps. Our definition identifies genes in a given species (human or mouse) with undetectable expression in same-species RNA but detectable expression in cross-species RNA. We demonstrate the comparative value of this method and discuss its potential contribution in cancer research.Results: Our method can identify genes from either species that demonstrate cross-hybridization and/or cross-alignment properties. Surprisingly, the set of genes identified using a simpler and more common approach (using a 'pure' cross-species sample and calling all detected genes as 'crossers') is not a superset of the genes identified using our technique. The observed levels of cross-hybridization are relatively low: 5.3% of human genes detected in mouse, and 3.5% of mouse genes detected in human. Observed levels of cross-alignment are practically comparable to the levels of cross-hybridization: 6.5% of human genes detected in mouse, and 2.3% of mouse genes detected in human. We also observed a relatively high percentage of orthologs: 40.3% of cross-hybridizing genes, and 32.2% of cross-aligning genes.Normalizing the gene catalog to use Consensus Coding Sequence (CCDS) IDs (Genome Res 19:1316-1323, 2009), our results show that the observed levels of cross-hybridization are low: 2.7% of human CCDS IDs are detected in mouse, and 2.4% of mouse CCDS IDs are detected in human. Levels of cross-alignment using the RNA-Seq data are comparable for the mouse, 2.2% of mouse CCDS IDs detected in human, and 9.9% of human CCDS IDs detected in mouse. However, the lists of cross-aligning/cross-hybridizing genes contain many that are of specific interest to oncologic researchers.Conclusions: The conservative definition that we propose identifies genes in mouse whose expression can be attributed to human RNA, and vice versa, as well as revealing genes with cross-alignment/cross-hybridization properties which could not be identified using a simpler but more established approach. The overall percentage of genes affected by cross-hybridization/cross-alignment is small, but includes genes that are of interest to oncologic researchers.

Original languageEnglish (US)
Article number8
JournalJournal of Clinical Bioinformatics
Issue number1
StatePublished - Apr 18 2013


  • Cross-alignment
  • Cross-hybridization
  • Microarray
  • Pathway analysis
  • RNA-Seq
  • Tumor microenvironment
  • Xenograft

ASJC Scopus subject areas

  • Health Informatics


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