In order to create species-specific primer pairs that detect only human mRNA sequences or only rat mRNA sequences within a human-rat cDNA library, the corresponding human and rat mRNA sequences must have a unique region of at least 60 bp or more. Using the human and corresponding rat FASTA mRNA sequences for EF1α, RPL13a and YWHAZ, we used Blast-n http://blast.ncbi.nlm.nih.gov/Blast.cgi to compare the sequences. EF1α had 99% sequence coverage (100% identity) between the human and rat mRNA sequences. RPL13a had 57% sequence coverage (87% identity) and YWHAZ transcript variants 1 and 2 had 92% - 63% sequence coverage (100% identity). Therefore, RPL13a and YWHAZ both were candidate human species-specific normalization genes while EF1α did not have a region containing a unique sequence (≥60 bp) in order to create primer pairs. Human species-specific primer pairs were constructed for the 2 normalization genes; RPL13a and YWHAZ and for 3 target genes; stanniocalcin-1 (STC-1), tumor necrosis factor, alpha-induced protein 6 (TSG6), and latent transforming growth factor binding protein 2 (LTBP2). NCBI Primer-BLAST http://www.ncbi.nlm.nih.gov/tools/primer-blast/ was used for primer pair sequence construction [23 ] using the species-unique mRNA sequences (FASTA format). Gradient PCR was used to determine optimum annealing temperature. All human and rat specific primer pairs were validated with RT-qPCR using cDNA from human MIAMI cells H3515(3) or rat hippocampal organotypic cultures either separately or in combination. All primer pairs produced 1 species-specific amplicon, with minimum off-target amplification. This was determined by the melting curve of each amplification reaction (Additional File # 2) and agarose gel electrophoresis (data not shown). Approximately 3-5 primer pairs were tested per human or rat species-specific normalization or target gene. All RT-qPCR results were normalized against a negative control, and the 2 normalization genes hRPL13a and hYWHAZ (human), or rRPL13a (rat). Using this same method rat specific primer pairs were also constructed for RPL13a, IGF1, IGFBP3, and IGFBP5.
Free full text: Click here