Advantages of Absolute Abundance Measurements
Whenever possible, measuring the abundance of cellular components (nucleic acids, proteins, and metabolites) on an absolute scale, e.g. number of transcripts per cell, is better than measuring relative levels, e.g. the ratio of a transcript in one sample versus another, is better because:

• it maintains the original quantitative data
• it permits direct comparison across multiple experiments, conditions, and laboratories
• it facilitates many types of analyses:
  • - construction of network models
  • - calculation of in vivo rate constants
  • - distinguishing between threshold- and gradient-based regulatory switches
  • - quantitation of transcription factor effects on promoter function
  • - determination of codon adaptation indices
  • - estimating translational efficiency
  • - performing cross-species comparisons

• loss of transcript abundance information in ratio data
• limited range of gene expression using data from a single scan
• inability to compare experiments that use different reference samples  

Our Calibrated Oligo Reference System
The figure below illustrates the difference between the conventional microarray protocol and our calibrated oligo reference system. In a conventional microarray experiment, two differentially labeled cDNA samples (Cy3 and Cy5) are co-hybridized to the same slide. The fluorescence intensity of each spot is measured by scanning (usually at a single scan intensity). Results are then expressed as a gene expression ratio between the two co-hybridized cDNA samples. In the calibrated oligo reference system, a cDNA sample is instead co-hybridized with a differentially labeled reference sample. The reference sample consists of a single oligo or mixture of oligos that are able to hybridize to each spot on the array. Following scanning at multiple scan intensities and processing through the masliner software to correct saturated values, the abundance of each transcript in the cDNA sample is expressed as a ratio to the oligo reference. Ratios between cDNA sample intensities and reference oligo intensities measure RNA levels on a scale that relates to their absolute abundance, instead of to the variable and unknown abundance of another cDNA sample.

We have demonstrated that results from the calibrated oligo reference system are accurate and retain absolute abundance information far better than conventional microarray ratios.

For more information see:

• Calibrated oligo references
• Masliner software
• Protocols
• The publication and supplementary Web site