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In the study and interpretation of expression profiles in tumor samples, the quantity and quality of the RNA and the heterogeneity of the tissue specimen from which it is extracted are both important. Optimization of amplification protocols has allowed researchers to use fewer tumor cells to obtain expression profiles for some clinical samples (1, 2). Assessment of RNA quality control has been improved by the use of highly sensitive gel analysis (3), and flow cytometry and laser microdissection techniques allow isolation of tumor cells from nondiseased connective tissue as well as inflammatory infiltrates (4,5). Several groups have reported the use of gene expression profiles from laser-microdissected tumors (6-8). However, the extent of the effect of the laser beam on the quality of the RNA and on the expression profile results remains unclear. Because the amount of RNA obtained after laser microdissection is generally low, opportunities for additional quality control and validation are limited (9,10). These issues led us to investigate the potential differences in gene expression profiles among samples obtained by different sample manipulation procedures. We compared the gene expression profiles of nonmicro-dissected (NE) samples with those from manually micro-dissected OCT blocks (OCT) and laser capture-micro-dissected sections (LASER; obtained with a PixCell microdissector) from aliquots of tumors obtained from the same patients. Four bladder transitional cell carcinomas and one breast carcinoma were evaluated. The expression profiles of the 15 tumor samples obtained with oligonucleotide HG-133A arrays containing 22 283 probes (Affymetrix) were analyzed by means of a t-test and hierarchical clustering (11,12). We estimated the effect of laser microdissection on gene profiling as well as potential sources of differences and criteria that may enable characterization of a sample as suitable for gene expression analysis. A detailed description of the materials and methods used and complementary information are provided as a Data Supplement available with the online version of this Technical Brief at http://www.clinchem.org/content/vo149/issue12/. We first evaluated the quality of the total RNA by means of absorbance ratios, gel analysis, and hybridization on a GeneChip test 3 array before hybridization on the HG-133A oligonucleotide arrays. The total RNA obtained from the 15 samples was sufficient (concentrations 0.1 [micro]g/[micro]L) and of adequate quality (based on [A.sub.260 nm/280 nm] ratios 1.8) for these analyses. We analyzed the gel images of the total RNA obtained by the Agilent 2100 Bioanalyzer for each sample (Fig. 1A) and observed that certain samples had similarly high total RNA quality, e.g., cases 2 and 102769. Conversely, other samples displayed lower total RNA quality, such samples 3- and 6-LASER or the three specimens from case 11. To assess the quality of the total RNA, we analyzed the characteristics of the probe obtained in the GeneChip test 3 array. The ratios of the signal intensities of the probe sets for [beta]-actin and glyceraldehyde-3-dehydrogenase (GAPDH) on the GeneChip test 3 arrays were used as hybridization controls (see the online Data Supplement). Samples 3- and 6-LASER, as well as the three samples from case 11, showed a 3'/5' ratio for GAPDH 5 and a 3'/5' ratio for [beta]-actin 65. This latter ratio was at least four times higher than the ratios for any of the 10 remaining specimens, suggesting that the extracted RNA was of poorer quality. For the LASER samples with poorer quality RNA (cases 3 and 6), the percentages of genes on the GeneChip were lower than for their corresponding OCT and NE aliquots. We found no remarkable differences in noise and background (web site), but the scaling factor was higher in the laser-microdissected samples compared with the nonmicrodissected samples, with means of 6.4 for the LASER samples, 4.3 for the OCT, and 4.7 for the NE (see the online Data Supplemen

Science & Nature
December 1
American Association for Clinical Chemistry, Inc.
The Gale Group, Inc., a Delaware corporation and an affiliate of Cengage Learning, Inc.

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