Due to the ever-increasing knowledge of intracellular signaling pathways, it is often desirable to examine multiple transcripts of interest within the same biological samples. This is taken to extremes in the case of micro-arrays, where it is now possible to examine the whole transcriptome from a single sample. qPCR retains its essential place in the canon of molecular techniques, as in the majority of studies, candidate genes are already known or implicated. The need to examine the expression of multiple transcripts is evident within several pathways within the eye, including photopigment complements, elements of the phototransduction cascade, inflammatory markers, markers of neuronal activity and clock genes. High-throughput approaches are therefore essential to face the rising number of candidates, making approaches based upon the construction of accurate exogenous controls cumbersome and rate-limiting.
The need to examine multiple transcripts places an effective limit on the number of genes which may be examined from any single sample. 1 pg of total RNA should provide enough cDNA for at least 20 target genes to be investigated, but with extended pathways of interest, the use of multiple internal controls or with low RNA yields, this can quickly become a limiting factor.
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