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RNArticles: RNA Amplification Used to Develop a New In Vitro HCMV Assay

Gene arrays can be used to simultaneously measure the expression levels of thousands of genes in a single experiment. One limitation of this technology is that large amounts of RNA may be required for each array hybridization experiment -- typically 10 g of total RNA. This becomes problematic when working with limited samples, such as small biopsies, mRNA-poor cells and tissues, primary cultured cells, and laser capture microdissection (LCM) samples, and, in this example, cells selected and collected by flow cytometry. RNA amplification is needed to perform array analysis on these limited samples.

Goodrum FD, Jordan CT, High K & Shenk T. (2002) Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor Cells: A model for latency. Proc Natl Acad Sci (USA) 99 (25): 16255-16260.

Ambion's MessageAmp aRNA Amplification Kit is based on an antisense RNA (aRNA) amplification procedure first described by Van Gelder and Eberwine (1). The protocol involves a series of enzymatic reactions resulting in linear amplification of exceedingly small amounts of RNA for use in array analysis. Here this method is applied to amplify RNA from a small number of human cytomegalovirus (HCMV) infected CD34+ progenitor cells to compare the expression pattern of these infected cells, an in vitro model, with that of normal fibroblasts and to correlate their expression with in vivo infected bone marrow cells.

HCMV is a ubiquitous herpes virus found in 80% of the population where it is maintained in a latent form. Infection of healthy normal individuals by HCMV is usually harmless, but reactivation of the latent virus in immune compromised individuals can be life threatening. The latent viral replication can be activated in a subset of bone marrow cells. Unfortunately, it is currently not possible to work with these cells in culture nor has there been an in vitro model for the study of the latent virus (3).

Recently, Goodrum et al. (2) used microarray analyses to establish the role of infected primary CD34+ haemotopoeitic progenitors grown above a murine stromal AFT024 monolayer as a potential in vitro model system to study HCMV latency. For this study, HCMV cDNA arrays comprised of PCR products from 222 known ORF's for the AD169 and Toledo strains of HCMV (2,4) were tested with cDNA probes made from primary human fibroblasts or human CD34+ progenitor cells infected in vitro with HCMV. RNA was isolated from 10,000-20,000 infected CD34+ cells and then amplified using Ambion's MessageAmp aRNA Amplification Kit. For the human fibroblasts, total RNA was isolated and DNase I treated with Ambion's DNA-free Kit. Poly(A) RNA selection was carried out using the Ambion MicroPoly(A)Pure Kit. cDNA probes were made by reverse transcription of the mRNA (for fibroblasts*) or amplified RNA (for CD34+ cells).

While all of the genes expressed in an active viral infection were observed with the fibroblasts during the complete course of viral replication (Figure 1, Panel A), the CD34+ cells showed expression of only a subset of viral genes during the 8 day time course following infection (Figure 1, Panel B). (Reactivation of viral replication was achieved in the CD34+ cells by
co-culturing with fibroblasts.) Analysis of the gene expression microarray data obtained from the latent CD34+ cells was validated by RT-PCR analysis. Thus the subset of genes expressed in the absence of active viral replication could be indicative of the subset of genes that are necessary for latent infection.

Figure 1. Comparison of HCMV Gene Expression in CD34+ and Primary Human Fibroblasts. (A) HCMV arrays were analyzed using cDNA probes made against primary human fibroblasts. (B) HCMV arrays were analyzed using cDNA probes against CD34+ cells. Each of the arrays has viral sequences spotted in triplicate, the bottom right most triplicate is spotted with cellular housekeeping genes as controls.

*Note: For the human fibroblast RNA, the gene expression profile did not vary significantly between amplified vs. unamplified samples.

We would like to thank Dr. Felicia Goodrum for pr oviding the data and for critical reading of the manuscript. Dr. Goodrum is a postdoctoral fellow in Dr. Thomas Shenk's laboratory at Princeton University.

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