By sequencing clones derived from RNA from different sources, and/or by normalizing cDNA libraries, the total set of genes sampled can be maximized.
Decreasing the prevalence of clones representing abundant transcripts by normalizing the cDNA before sequencing may significantly increase the efficiency of random sequencing and is essential for rare gene discovery [ 22, 23].
Furthermore, we increased the representation of genes expressed in low-copy number by normalizing our amplified cDNA using a double-stranded nuclease (Trimmer-Direct Kit; Evrogen, Moscow).
The relative copy numbers of mRNAs were calculated by normalizing cDNAs to 28S rRNA using the Comparative Quantitation module of the Rotor-Gene 6000 software (Version 1.7.28, Corbett Research), which automatically calculates the real-time PCR efficiency sample-by-sample.
Gene expression was quantified by normalizing the logarithmic cycle threshold (CT) value (2−CT) to the cDNA content of each sample to obtain the expression 2−CT/cDNA content (ng/ml).
For every transcript, each cDNA sample was analyzed in triplicate, and relative transcript abundance was calculated by normalizing to the maximum level.
For every transcript, each cDNA sample was analyzed in triplicate, and relative transcript abundance was calculated by normalizing to the maximum amount of concentration.
Applying the formula CTgene−CTGapdh = ΔCT normalized each gene to measurements for Gapdh cDNA in the same sample.
We used three genes, that is hEEF1A1, hGAPDH and hHRPT1, to normalize input cDNA for each sample, with cDNA of normal tissue used as calibrator.
cDNA synthesis was carried out using the SMART cDNA construction kit (Clontech) and normalized using a cDNA Normalization Kit (Evrogen) by the Dragon Genomics Centre, TAKARA BIO Inc. (Yokkaichi, Japan).
