Simultaneous isolation of DNA and RNA from the same cell population obtained by laser capture microdissection for genome and transcriptome profiling.

Laser capture microdissection (LCM) is used extensively for genome and transcriptome profiling. Traditionally, however, DNA and RNA are purified from separate populations of LCM-harvested cells, limiting the strength of inferences about the relationship between gene expression and gene sequence variation. There have been no published protocols for the simultaneous isolation of DNA and RNA from the same cells that are obtained by LCM of patient tissue specimens. Here we report an adaptation of the Qiagen AllPrep method that allows the purification of DNA and RNA from the same LCM-harvested cells. We compared DNA and RNA purified by the QIAamp DNA Micro kit and the PicoPure RNA Isolation kit, respectively, from LCM-collected cells from adjacent tissue sections of the same specimen. The adapted method yields 90% of DNA and 38% of RNA compared with the individual methods. When tested with the GeneChip 250K Nsp Array, the concordance rate of the single nucleotide polymorphism heterozygosity calls was 98%. When tested with the GeneChip U133 Plus 2.0 Array, the correlation coefficient of the raw gene expression was 97%. Thus, we developed a method to obtain both DNA and RNA material from a single population of LCM-harvested cells and herein discuss the strengths and limitations of this methodology.

Identification of radiation-induced expression changes in nonimmortalized human T cells.

In the event of a radiation accident or attack, it will be imperative to quickly assess the amount of radiation exposure to accurately triage victims for appropriate care. RNA-based radiation dosimetry assays offer the potential to rapidly screen thousands of individuals in an efficient and cost-effective manner. However, prior to the development of these assays, it will be critical to identify those genes that will be most useful to delineate different radiation doses. Using global expression profiling, we examined expression changes in nonimmortalized T cells across a wide range of doses (0.15-12 Gy). Because many radiation responses are highly dependent on time, expression changes were examined at three different times (3, 8, and 24 h). Analyses identified 61, 512 and 1310 genes with significant linear dose-dependent expression changes at 3, 8 and 24 h, respectively. Using a stepwise regression procedure, a model was developed to estimate in vitro radiation exposures using the expression of three genes (CDKN1A, PSRC1 and TNFSF4) and validated in an independent test set with 86% accuracy. These findings suggest that RNA-based expression assays for a small subset of genes can be employed to develop clinical biodosimetry assays to be used in assessments of radiation exposure and toxicity.

IL10 and IL10 receptor gene variation and outcomes after unrelated and related hematopoietic cell transplantation.

BACKGROUND: Results of a previous study with human leukocyte antigen (HLA)-identical siblings showed individual and synergistic associations of single nucleotide polymorphisms in the promoter region of the recipient's IL10 gene and the donor's IL10 receptor beta (IL-10RB) gene with development of grades III-IV acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation. METHODS: In this study of 936 patients who had unrelated donors, genotypes of single nucleotide polymorphisms in the IL10 gene and the IL-10RB gene were evaluated as correlates with outcomes after transplantation. RESULTS: We found no statistically significant associations of polymorphisms at positions -3575, -2763, -1082, and -592 of the IL10 gene or codon 238 of the IL10RB gene with severe acute GVHD, extensive chronic GVHD or nonrelapse mortality after hematopoietic cell transplantation. Among HLA-matched unrelated pairs, the patient's IL10/-592 genotype and donor's IL10RB/c238 genotype showed trends suggesting individual and combined associations with grades III-IV acute GVHD similar to those observed among patients with HLA-identical sibling donors. CONCLUSIONS: Although genetic variation in IL10 pathway affects risk of acute GVHD and non-relapse mortality in HLA-identical sibling transplants, the current results indicate that genetic variation in the IL10 pathway does not significant affect these outcomes in unrelated donor transplants suggesting that the strength of the alloimmune response in the latter exceeds the anti-inflammatory activity of IL10.

Genetic variation in the IL-10 pathway modulates severity of acute graft-versus-host disease following hematopoietic cell transplantation: synergism between IL-10 genotype of patient and IL-10 receptor beta genotype of donor.

We have previously shown that the interleukin 10 (IL-10)/-592*A allele of the recipient is associated with less severe acute graft-versus-host disease (GVHD) and a lower risk of nonrelapse mortality after hematopoietic cell transplantation (HCT) from an HLA-identical sibling. In the present study, we examined variation in the IL-10 receptor beta gene as a further test of the hypothesis that the IL-10 pathway regulates the risk of acute GVHD. A single nucleotide polymorphism (A/G) at cDNA position 238 of the IL-10 receptor beta gene (IL10RB/c238) was genotyped in 953 HC transplant recipients and their HLA-identical sibling donors. IL-10/-592 and IL10RB/c238 genotypes were tested for association with GVHD by multivariable analysis. The IL-10/-592*A allele of the recipient and IL10RB/c238*G allele of the donor were significantly associated with a lower risk of grades III-IV acute GVHD (trend P < .001 and P = .02, respectively). The donor IL10RB/c238*G allele provided protection among patients with the IL-10/-592 A/C or A/A genotypes but not among patients with the high-risk IL-10/-592 C/C genotype. These data suggest an interaction of the patient IL-10/-592 and donor IL10RB/c238 genotypes on risk of GVHD, further supporting the hypothesis that the IL-10 pathway plays an important role in controlling the severity of acute GVHD.