The abundance of each transcript was then normalized by calculating the transcript abundance in Fragment Per Kilobase of exon per million fragments mapped (FPKM).
In order to calculate the transcript level ratios, it was assumed that each PCR cycle results in exact doubling of the amounts of amplicons.
What does it mean to calculate the transcript abundance of a gene at an expression level of 50 or of 200?
To examine the consequences of this approach on the sequencing of highly expressed transcripts, the relative expression level of unigenes was estimated by calculating the transcript abundance expressed as reads per kilobase per million mapped reads (RPKM) [ 26].
For each oligonucleotide primer pair and every plate, a standard curve was created from genomic DNA derived from the corresponding cell line, thereby calculating the transcript copy numbers relative to the genomic locus copy number.
All amplification reactions were performed in triplicate, and the averages of the threshold cycles were used to interpolate standard curves and to calculate the transcript amount in samples using SDS version 1.7a software.
The slides were scanned by an Agilent G2566AA scanner (Agilent Technologies, Inc., Santa Clara, CA) at two PMT settings of 100 and 50, enabling correction of saturated spot intensities [ 35] and estimation of the scanner amplification factors needed in TransCount to calculate the transcript concentrations [ 10].
To account for potential bias introduced by using only uniquely mapped reads, Cuffdiff v2.2.1 (Trapnell et al. 2013) (which incorporates nonuniquely mapped reads as well) was used as an alternative approach to calculate the transcript abundances and fold changes and the results were highly similar (supplementary fig. S3 and table S10, Supplementary Material online).
We calculated the pairwise transcript correlations within each intersected module or men and women separately.
To contextualize these newly annotated ORs, IRs, and GRs within the olfactory tissues of T. amboinensis, we next calculated the relative transcript abundance levels in terms of RPKM.
