Purified DNA was used as templates for qPCR quantification using Tbg or Lpl specific primers listed in Table 2.
Purified RNAs were treated with RNase-free DNAse (DNA-free, Ambion) and reverse-transcribed, for set 1, with Superscript II (Invitrogen) and, for set 2, Omniscript (Qiagen) to generate the corresponding cDNAs that served as PCR templates for mRNA quantification.
Purified RNAs were treated with RNase-free DNAse (DNA-free, Ambion) and reverse-transcribed, with Superscript II (Invitrogen) to generate the corresponding cDNAs that served as PCR templates for mRNA quantification.
cDNA was generated from 1 ug of RNA using Omniscript reverse transcriptase (Qiagen) and oligo-dT primers to generate template for GAPDH quantification and an ORF50-specific reverse primer (5' GGAGAGGGACTACTGTTGG 3') to generate template for ORF50 mRNA quantification.
The resulting cDNA products were dissolved in a final volume of 200 μL, and a 2-μL aliquot was used as template for subsequent quantification by real-time PCR amplification.
Out of eighteen question templates, four templates for the question types Quantification and Concept Comparison were only used for main concepts, for which the system could find at least two related hyponyms on WordNet.
The switch between the exponential and the linear phases of LATE-PCR occurs at the CT, independently for each template, so that template quantification is achieved exactly as in symmetric PCR.
The one-step RT-PCR was started with cDNA synthesis at 50°C for 30 min and denaturation step at 94°C for 2 min followed by 30 PCR cycles of denaturation at 94°C for 15 s, annealing at 50°C for 30 s and extension at 68°C for 90 s and a final extension step at 68°C for 5 min. A second nested PCR was used for template quantification and in some of the control experiments.
Remaining RNA was used for template quantification (see below).
For each genome fragment, both outer PCR primers and nested, inner primers were designed; inner primers were only used for template quantification and internal testing purposes.
