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APPLICATION
Human Rapid-Scan Gene Expression Panel The Human Rapid-Scan Gene Expression Panel consists of cDNAs derived from 24 human tissues. The cDNAs were arrayed in a 96-well PCR plate (Fig. 1) and the panel used to analyze transcript accumulation for a series of control genes. The products of Rapid-Scan PCR were separated in an agarose gel and stained with ethidium bromide (Fig. 2). To facilitate comparison of transcript accumulation in different tissues, first-strand cDNAs are normalized using actin as an internal control. Amplification of actin cDNA (using primers provided) shows equivalent distribution in all 24 human tissues. The Human Rapid-Scan Gene Expression Panel has been used to determine the relative accumulation of the muscle-specific a-actin 2 transcript, which is detected predominantly in skeletal muscle and heart, but also in brain. This panel may also be used to quantify relative levels of alternatively spliced transcripts in various tissues or differentiation states. The APP695 transcript differs from the APP770 transcript by a 225-bp (75 amino acids) deletion, due to alternate RNA splicing. While APP770 was detected in all adult tissues, it was not present in the two fetal tissues. In contrast, APP695 was detected in only fetal brain and adult brain. Profiling of carbonic anhydrase expression using a Rapid-Scan panel confirmed its presence in colon, as well as bone marrow. Analysis of the prostate-specific NKX3.1 gene, a homeo-domain containing transcriptional factor, revealed expression in testis, salivary gland and PBL, at levels almost two logs below that in prostate.
Fig 1 and 2 Human Brain Rapid-Scan Gene Expression Panel The Human Brain Rapid-Scan Gene Expression Panel consists of cDNAs derived from 12 human brain parts. RNA was prepared and pooled from two individuals and cDNAs were arrayed in a 48-well PCR plate in the order indicated (Fig. 3). The Human Brain Rapid-Scan Gene Expression Panel was used to determine the relative transcript levels of a gene family. Three members of the human dopamine receptor family were analyzed and the PCR products were separated in an agarose gel (Fig. 4). The D5 dopamine receptor was present at more or less equal levels in all of the brain parts examined. The D2 receptor also showed broad distribution but at varying levels of accumulation. Alternatively, the D1 receptor was only expressed in the caudate nucleus, verifying results that have been previously observed. The results also demonstrate equivalent levels of actin in each normalized tissue.
Fig 3 and 4 Human Breast Cancer Rapid-Scan Gene Expression Panel The Human Breast Cancer Rapid-Scan Gene Expression Panel consists of cDNAs derived from 12-normal and 12-tumor breast tissues. Normal tissues were obtained by reduction mammaplasty and micro dissected to remove fat tissues. Tumor tissues were from patients with carcinoma of the breast. The cDNAs were then arrayed in a 96-well PCR plate in the order indicated (Fig. 5). The Human Breast Cancer Rapid-Scan Gene Expression Panel was designed to examine the change in gene expression of a candidate gene between normal and tumor tissues. To illustrate, the expression of two new oncogenes (Gene 1 and 2) and one previously reported breast cancer related gene (MRG) were examined using this panel. Transcripts of gene I and 2 were undetectable in normal breast tissues but were up regulated in many of the breast tumor tissues. In contrast, the expression of the MRG (mammary derived growth inhibitor related) gene was detected in all 12 normal breast tissues, but undetectable in 5 of 12 tumor tissues (Fig 6).
Fig 5 and 6 Mouse Rapid-Scan Gene Expression Panel The Mouse Rapid-Scan Gene Expression Panel consists of cDNAs derived from 24 different tissues or developmental stages. Adult tissues were obtained from outbred Swiss Webster mice, and the breast tissues from outbred CD1 mice. The cDNAs were normalized against actin and The Mouse Rapid-Scan Gene Expression Panel was used to obtain the expression profile of an uncharacterized EST or to confirm tissue-specific expression. A nominal skin-specific EST sequence was expressed in skin, as expected, and also in stomach (Fig 8).
Fig 7 and 8 Mouse Brain Rapid-Scan Gene Expression Panel The Mouse Brain Rapid-Scan Gene Expression Panel consists of cDNAs derived from 48 mouse brain parts at five developmental stages. Brain tissues were harvested from Outbred NIH Swiss mice. The cDNAs were normalized against actin and arrayed in a 96-well PCR plate in the order indicated (Fig. 9). The Mouse Brain Rapid-Scan Gene Expression Panel was used to profile the expression of mouse DLX-5 (Drosophila homolog Distal-less 5), dopamine receptor DD3, and Hox3.1. In embryonic stages, Hox3.1 is expressed in many brain parts, with a high level of expression in the spinal cord. In adults, however, Hox3.1 expression is restricted to the spinal cord (Fig. 10).
Fig 9 and 10 Drosophila Tissue Rapid-Scan Panel The Drosophila Rapid-Scan Gene Expression Panel consists of cDNAs derived from 12 different tissues and developmental stages. RNA was prepared from Drosophila melanogaster Canton S strain and cDNAs arrayed in a 48-well PCR plate in the order indicated (Fig. 11). The Drosophila Rapid-Scan Gene Expression Panel was used to confirm the expression profiles of genes whose sites of expression have already been established. The expression of the actin genes in Drosophila is developmentally regulated, therefore rp49, and -the gene encoding a ribosomal protein - was used as an internal control to normalize each Drosophila cDNA. The male-specific Fruitless transcript was detected only in the adult male head. The alternatively spliced male form of Doublesex was detected mainly in the 3rd instar and male tissues, with no expression indicated in female tissues. The Eyeless transcript was detected in whole embryos, (starting at 4 hours) and in adult head, but not in adult body (Fig 12).
Fig 11 and 12
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