A critical element in using cDNA for gene discovery or expression analysis is the quality of the cDNA. Investigators often presume that the distribution and abundance of mRNAs are maintained in the cDNAs and the libraries made from them; many criteria are of concern (e.g., complete representation, percentage of full-length inserts, lack of ribosomal sequences, lack of mitochondrial/yeast/E. coli sequences, percentage of colonies with inserts, percentage of colonies yielding good sequence).
Typically, one or possibly two features of the cDNA are addressed: representation and cDNA length. To examine these issues, RT-PCR or probe signals derived from traditional housekeeping genes are evaluated. Unfortunately, transcripts for housekeeping genes are abundant, and suboptimal library construction can still yield a strong signal through RT-PCR or by probing. In gene expression analysis, housekeeping genes are typically expressed at higher levels than the gene of interest, which makes comparisons and quantification difficult.
To better analyze library quality, take into account the abundance of the
target gene and the placement of RT-PCR primers or probe along the gene
transcript, so both representation and insert size can be addressed. With
respect to these discovery technologies, representat