Inferring the gene expression status of patients’ tumors via analysis of the residual tumor material is often challenged by the small quantity of tumor cells or tissues available. Recent technological advances in cDNA synthesis, whole transcriptome amplification, and library construction make it possible to routinely analyze transcriptomes from picogram-level of input RNA.
At IQVIA Laboratories we are constantly looking for new ways to improve the methods we utilize in our lab. We have recently modified the conventional SMART-seq® protocol to incorporate a library size selection procedure.
This modification improved the ease in which the libraries could be normalized to equimolar fractions and thus to cluster much more predictable on the flow cell. This modification represents a methodological improvement of transcriptomic analysis from small quantities of total RNA. We also addressed concerns that the modified steps might introduce serious biases, invalidating the existing data analysis approaches. Compared to previous versions of the SMARTer method, the modified SMART-Seq v4 procedure implemented at IQVIA Laboratories significantly improved the percentage of reads aligned to the transcriptome, as well as the total number of genes detected, all while reducing technical variability.
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