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

href="http://www.ambion.com/catalog/CatNum.php?1918">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
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