Presented at:
XXIIth International Congress of Immunology, Montreal, QC, Canada, July 18-23, 2004
Abstract #471
Comparison of new standard and amplification procedures with GeneChip arrays for studying T cell activation
Zhu S, Wong L, Hu D, Heinemeyer E, Huntsman S, Boden E, Chikuma S, Ye J, Seyfert-Margolis V, Bluestone JA
Diabetes Center, University of California, San Francisco and Immune Tolerance Network, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA.
GeneChip technology allows for gene expression profiling of the entire genome using a variety of biological materials. However, Affymetrix standard protocols require a minimum of five micrograms of total RNA as starting material. There are many instances where only a limited amount of RNA can be obtained for analysis by GeneChips, including laser capture micro-dissection, sorted cells from tissue or blood, or small biopsy samples from patients. Such sparse starting material remains a major limiting factor in gene expression profiling.
We have developed a reproducible new standard procedure (one run) that reduces the RNA starting material to 1 m g of total RNA, without compromising data quality. We have also established a new amplification (two run) method that requires as little as 1 ng of total RNA as starting material. A gene expression comparison was performed with human Jurkat cells stimulated with anti-CD3 antibody, in an effort to identify genes participating in T cell activation using the standard (2.5 m g RNA) and amplification (50 ng RNA) procedures for GeneChip arrays. Statistical analysis, with the cut-off at p<0.05 and fold change >2.0, identified 116 genes by the standard procedure and 119 genes by the amplification procedure. Greater than 60% of significant genes shared identity by both procedures. Quantitative Taqman RT-PCR (Q-PCR) was performed with 30 selected genes to validate their expression during T cell activation as measured with GeneChip arrays. The 30 genes were selected based on identification by: both procedures (n=10); the standard procedure alone (n=10) and the amplification procedure alone (n=10). We observed that the expression levels of 24 genes were similar by both methods while 6 genes exhibited higher fold changes by Q-PCR. Coefficients of variation for each gene, associated with triplicate measurements by Q-PCR, ranged from 0.48 to 0.006 percent. Some genes previously reported to be associated with T cell activation, such as ICOS, CCR2, CD45, EMP1, and CLACA1, were identified by the amplification procedures but not by the standard procedure. However, other T cell activation-related genes, including BCL6, CD6, CD40L, IL16 and CD96, were identified by the standard procedure but not by the amplification procedure.
In conclusion, it is now possible to screen expression profiles with a low quantity of starting material, using GeneChip arrays. However, the different methods can identify unique gene sets that may have biological relevance in various research and clinical settings.