Quality-Controlled Measurement Methods for Quantification of Variations in Transcript Abundance in Whole Blood Samples from Healthy Volunteers

Abstract

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

Interindividual and Interethnic Variation in Genomewide Gene Expression: Insights into the Biological Variation of Gene Expression and Clinical Implications

“…The same conclusion was reached recently in an evaluation of QC parameters in a setting of clinical laboratory tests (19 ). Almost half (43%) of the total variation was due to interindividual variation.…”

“…Recently, Peters et al examined interindividual and intraindividual variation in gene expression in blood samples (19 ). They used standardized reverse transcription-PCR analysis to measure the expression of 19 marker genes.…”

“…Interindividual variation accounted for almost half of the total variance and was the major component of biological variation; however, the magnitude of the interindividual variation in these 19 marker genes varied over a 4-fold range, with CVs ranging from 10% to 40%. Markers with lower interindividual variation were more informative than those with high variation and had a higher individuality index (17,19 ).…”

Quantification of Drug-Induced mRNA in Human Whole Blood ex vivo

“…After incubation of PAXgene tubes at room temperature for 2 hours, the samples were stored frozen at −80 °C. The samples were shipped to Asuragen (Austin, TX) for total RNA purifi cation, similar to the recent publication (Peters et al 2007). The cDNA was then synthesized in solution by the standard protocol (Promega) using either oligo(dT) or random hexamers as primers.…”

“…The next challenge is to identify abnormal levels of mRNA that commonly exist in both normal and disease states. Although normal reference values of these mRNA were quantifi ed using healthy subject population (MAQC Consortium, 2006;Mitsuhashi et al 2006;Peters et al 2007;Zheng et al 2006), the identifi cation of disease-specifi c mRNA levels is one of the current topics in molecular diagnostics development.…”