RNA extraction for arrays.

DNA microarrays enable insights into global gene expression by capturing a snapshot of cellular expression levels at the time of sample collection. Careful RNA handling and extraction are required to preserve this information properly, ensure sample-to-sample reproducibility, and limit unwanted technical variation in experimental data. This chapter discusses important considerations for "array-friendly" sample handling and processing from biosamples such as blood, formalin-fixed, paraffin-embedded samples, and fresh or flash-frozen tissues and cells. It also provides guidelines on RNA quality assessments, which can be used to validate sample preparation and maximize recovery of relevant biological information.

The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs.

BACKGROUND: Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields. RESULTS: We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems. CONCLUSIONS: This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site.

Quality-controlled measurement methods for quantification of variations in transcript abundance in whole blood samples from healthy volunteers.

BACKGROUND: Transcript abundance (TA) measurement in whole blood frequently is conducted to identify potential biomarkers for disease risk and to predict or monitor drug response. Potential biomarkers discovered in this way must be validated by quantitative technology. In this study we assessed the use of standardized reverse transcription PCR (StaRT-PCR) to validate potential biomarkers discovered through whole blood TA profiling. METHODS: For each of 15 healthy volunteers, 6 blood samples were obtained, including 3 samples at each of 2 separate visits. Total variation in TA for each gene was partitioned into replicate, sample, visit, study participant, and residual components. RESULTS: Variation originating from technical processing was <5% of total combined variation and was primarily preanalytical. Interindividual biological sample variation was larger than technical variation. For 12 of 19 tests, the distribution of measured values was gaussian (Shapiro-Wilks test). CONCLUSION: For control or diseased population groups with variation rates as low as those observed in this control group, 17 individuals per group would be required to detect 1 SD change with 80% power with a 2-sided alpha = 0.05 statistical test for mean differences.