Transcript abundance measurement
Total RNA samples extracted from NBEC were reverse transcribed using M-MLV reverse transcriptase and oligo dT primers as previously described [9,11]. Standardized RT (StaRT)-PCR was used for transcript abundance measurement in these studies. With StaRT-PCR, an internal standard for each gene within a standardized mixture of internal standards (SMIS) is included in each PCR reaction. After amplification, products were electrophoresed on an Agilent 2100 Bioanalyzer using DNA Chips with DNA 1000 Kit reagents for visualization according to the manufacturer's protocol (Agilent Technologies Deutschland GmbH, Waldbronn, Germany).
The StaRT-PCR technology is licensed to Gene Express, Inc. (Toledo, OH). Many of the reagents are available commercially and were obtained through Gene Express, Inc. for this study. StaRT-PCR reagents for each of the measured genes that were not commercially available, including primers and SMIS, were prepared according to previously described methods [11,12]. Sequence information for the primers is provided in Table 2.
Including an internal standard within a SMIS in each measurement controls for all known sources of variation during PCR, including inhibitors in samples, and generates virtually-multiplexed transcript abundance data that are directly comparable across multiple experiments and institutions [13]. The performance characteristics of StaRT-PCR are superior to other forms of commercially available quantitative PCR technology in the areas critical to this study. With respect to these studies, the key property of a quantitative PCR method is not whether the PCR products are measured kinetically or at endpoint, but rather whether there are internal standards in each measurement or not. The overall performance characteristics of StaRT-PCR, including extensive validation of the method in independent laboratories have been presented in several recent articles and chapters [13-15]. With respect to the genes measured in this study, for each gene the StaRT-PCR reagents had lower detection threshold of less than 10 molecules, linear dynamic range of more than six orders of magnitude (less than 10 to over 107 molecules), and signal-to-analyte response of 100%. In addition, the presence of an internal standard controls for inter-sample variation in presence of PCR inhibitors (which often are gene-specific) and ensures no false negatives (if the PCR fails the internal standard PCR product is not observed and there are no data to report). False positives are eliminated through use of a control PCR reaction with no cDNA in it.