Quality assessment of cross‐species hybridization of CHO transcriptome on a mouse DNA oligo microarray

Abstract

DNA microarray technology has been widely utilized for species with extensive genome sequence information available. Given the limited genomic information pertaining to Chinese hamster ovary (CHO) cell line, cross-species hybridization using mouse microarrays provides a viable alternative. In this study, the utility of mouse Affymetrix microarrays for transcriptome profiling in CHO cells was assessed by hybridizing identical sets of cRNA samples from CHO cells on both mouse and CHO Affymetrix microarrays. Expression level measured by probe sets for orthologous transcripts on the two microarrays was compared. Only a fraction of the orthologous probes which detected expression calls in same species hybridization were similarly called present in cross species hybridization. In further analysis at the 25-mer probe level, it was revealed that specific hybridization signals were detectable by the subset of mouse probes that have a high degree of homology to the corresponding CHO sequences. The feasibility of cross species hybridization for quantifying the extent of differential expression was assessed by comparing transcript levels of CHO cells cultivated with and without sodium butyrate. While same species hybridization gave consistent degree of differential expression calls in replicated runs, a much inferior ability in quantifying differential expression was seen with cross species hybridization. Our results demonstrate that through detailed analysis of homology at the probe pair level, a subset of probes on existing mouse Affymetrix oligo-array can be used successfully for transcriptome profiling of CHO cells.

CHO gene expression profiling in biopharmaceutical process analysis and design

“…Harvested cells (2 Â 10 6 cells) were centrifuged and the total RNA was extracted from the cell pellets using the RNeasy Kit (Qiagen GmbH, Hilden, Germany) according to manufacturer's protocol. Ten micrograms of the extracted biotin labeled cRNA was hybridized on a custom-made CHO-specific Affymetrix microarray (Yee et al, 2008b) containing 10,118 probe sets with 7,525 unique sequences that represent a subset from a collection of 16,136 unique CHO cell and Chinese hamster transcripts obtained by comprehensive EST sequencing as described by Wlaschin et al (2005) and Wlaschin and Hu (2007). Samples were scanned in highresolution mode and data were processed with an Affymetrix GeneChip Scanner 3000 system (Affymetrix, Inc., Santa Clara, CA).…”

“…In this study, gene expression profiling was performed in an IgG producing CHO cell line cultivated in industrial fed batch process formats using a CHO-specific Affymetrix microarray (Wlaschin et al, 2005;Yee et al, 2008b) to assess the potential use of this technology in biopharmaceutical process development. Specifically, we investigated the number and extent of gene expression changes during the time course of a representative HT and a LT fed batch process applying two different nutritional regimes.…”

Development and characterization of a Chinese hamster ovary cell-specific oligonucleotide microarray

“…A small number of independent efforts have lead to development of similar tools, most notably the Consortium for CHO Cell Genomics (headed by the laboratory of Wei-Shou Hu at the University of Minnesota, Minneapolis, MN), where sequence mining led to the development of a cDNA microarray platform (Wlaschin et al 2005), and more recently a microarray constructed on the Affymetrix platform (Kantardjieff et al 2009). Several studies in this 3 area have attempted to utilize commercial microarrays from related species, such as mouse, with some success (Ernst et al 2006;Yee et al 2008). However, while there may be applications where this approach is applicable, we demonstrate here that a more specific tool is necessary when one is attempting to dissect alterations in specific, individual genes.…”

Functional Analysis of the Gossypium arboreum Genome

“…Hybridization in these studies is necessarily broadened to include imperfect matches and has a higher likelihood of including poorly related genes (Koltai and Weingarten-Baror 2008). Nonetheless, cross-species microarray studies have been demonstrated repeatedly to be valuable for study of species that have no microarrays available and/or have very limited sequencing or annotation available (Kassahn 2008;Thayer et al 2008;Yee et al 2008). In our studies, lower levels of hybridization not only result largely from lower homologies between the target and probe sequences but may also result from differences in gene copy number (Kassahn 2008).…”