Comparison of Amplification Methods to Produce Affymetrix GeneChip Target Material

Details

• Creators:
  Dennis, Burian ; White, Vicky ; Huggins, Mark ; Kupfer, Doris M. ; Canfield, Dennis V. ; Whinnery, James E.
• Corporate Creators:
  United States. Department of Transportation. Federal Aviation Administration. Office of Aviation. Civil Aerospace Medical Institute
• Corporate Contributors:
  United States. Department of Transportation. Federal Aviation Administration. Office of Aviation. Office of Aerospace Medicine
• Subject/TRT Terms:
  • Experiments
  • Quality assurance
  • Sensitivity analysis
  • Statistical analysis
  • Microarray Analysis
  • Affymetrix GeneChip®
  • RNA Amplification
  • Globin-RNA Reduction
  • Whole Blood
• Publication/ Report Number:
  DOT/FAA/AM-07/9
• Resource Type:
  Tech Report
• Right Statement:
  Public Domain
• Geographical Coverage:
  United States
• Corporate Publisher:
  United States. Department of Transportation. Federal Aviation Administration. Office of Aviation. Civil Aerospace Medical Institute
• Abstract:
  Whole blood from living subjects is a convenient matrix to use as a source of RNA for microarray experiments with human subjects especially when subject material is collected at a location other than the collaborating site conducting the microarray work. Collection methods for whole blood that include stabilization of the RNA are known but suffer from issues of decreased sensitivity due to the large amount of globin RNA present from reticulocyte lysis. The experiments presented here were designed to test a globin-RNA reduction protocol in conjunction with three different amplification methods. Statistical analysis of the six different protocols, coupled with post-hybridization quality assurance methods, revealed that an amplification protocol that yielded a fragmented biotin-labeled cDNA product resulted in the highest Percent Present calls from the Affymetrix analysis software and the least methodology based variability. Based on these results, this amplification protocol is expected to lead to the greatest sensitivity and accuracy for differential expression testing of the six amplification methods tested.
• Format:
  PDF
• Download URL:
  https://rosap.ntl.bts.gov/view/dot/58449/dot_58449_DS1.pdf
• File Type:
  [PDF - 594.21 KB]
• Collection(s):
  FAA Civil Aerospace Medical Institute
• Main Document Checksum:
  urn:sha256:30a92680a3583ae07282bdd19731105b2a532d6bbc8ebb8189af2f618e5258e1