title: A highly reproducible, linear, and automated sample preparation method for DNA microarrays.
abstract: DNA microarrays are powerful tools to detect changes in transcript abundance in multiple samples in parallel. However, detection of differential transcript levels requires a reproducible sample (target) preparation method in addition to a high-performance microarray. Therefore, we optimized a target-preparation method that converts the poly(A)(+) RNA fraction of total RNA into complementary DNA, then generates biotin-labeled complementary RNA from the cDNA. We measured the efficiency of incorporation of biotin-containing nucleotides by an enzymatic digestion, followed by resolution via analytical high-performance liquid chromatography (HPLC). When the target was hybridized to a sensitive and reproducible microarray platform, low coefficients of variation in both hybridization intensities and differential expression ratios across target preparations were observed. Nearly identical hybridization intensities and expression ratios are observed regardless of whether poly(A)(+)-enriched RNA or total RNA is used as the starting material. We show the ability to discern biological and production variability through the use of different lots of commercial samples as visualized by hierarchical clustering. Automation of the target-preparation procedure shows equivalence to the manual procedure, reproducible yields of target, and low variability as measured by hybridization to microarrays. Most importantly, RNA mixing experiments show a linear and quantitative amplification in probe hybridization signals for >6000 genes across the entire signal range.
authors.affiliation: Motorola Life Sciences, Northbrook, IL 60062, USA. David.Dorris@email.mot.com
authors.forename: David R
authors.initials: DR
authors.lastname: Dorris
authors.forename: Ramesh
authors.initials: R
authors.lastname: Ramakrishnan
authors.forename: Dionisios
authors.initials: D
authors.lastname: Trakas
authors.forename: Frank
authors.initials: F
authors.lastname: Dudzik
authors.forename: Richard
authors.initials: R
authors.lastname: Belval
authors.forename: Connie
authors.initials: C
authors.lastname: Zhao
authors.forename: Allen
authors.initials: A
authors.lastname: Nguyen
authors.forename: Marc
authors.initials: M
authors.lastname: Domanus
authors.forename: Abhijit
authors.initials: A
authors.lastname: Mazumder
mesh_terms: D019921:Biotinylation; D002051:Burkitt Lymphoma / Q000473:pathology; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D006801:Humans; D016014:Linear Models; D008113:Liver Neoplasms / Q000473:pathology; D021141:Nucleic Acid Amplification Techniques; D009711:Nucleotides / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D009711:Nucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012313:RNA