Highly sensitive and quantitative whole transcriptome and surrogate transcriptome targeted sequencing TempO-Seq assays were used to profile gene expression from 1 mm sq focal areas of FFPE sections (5 μm thick) of normal and cancerous tissue within the same prostate. The whole transcriptome assay directly measures changes within the whole transcriptome. The surrogate assay targets fewer genes yet permits in silico extrapolation to identify changes across the whole transcriptome. Both approaches identified differentiating biomarkers and mechanistic pathways in prostate cancer, both corroborating what has been reported and adding new mechanistic insights which will be discussed. These new observations likely result from the sensitivity of the TempO-Seq assays and their robust performance measuring gene expression from focal areas of FFPE, which in turn permit improved isolation of histology. Benefits derived from the TempO-Seq platform which will be discussed are:

1) Insensitivity to RNA degradation (measurements from intact RNA RIN = 9.1 correlate to measurements from degraded RNA RIN = 3.0, R2 = 0.97).

2) Does not require RNA extraction. Measures total RNA from FFPE samples, both soluble RNA in the lysate and crosslinked insoluble RNA.

3) Simple capture-free ligation-based assay protocol, without 3’ or 5’ bias to the probes used to measure each gene. No requirement for poly-adenylation or capture of the RNA (typically required in ligation-based assays).

4) Single base specificity derived from the specificity of probe ligation.

5) Misligation rates <0.01% (unlike other ligation-based assays where misligation can be as great as 30%) enable no sample background controls to be run with typical counts of 0 for most genes and 1 to 3 counts for a few genes.

6) High signal-to-noise provides single cell sensitivity and ability to measure differential gene expression from small focal areas of tissue.

7) Excellent reproducibility (average ∼5% CV) between biological replicates across all expressed genes results in high significance of even small fold change differences.

8) Sequencing only the ligated probes complementary to a 50-base sequence of each gene eliminates the need for bioinformatics to analyze the sequencing data. Pooling of samples into a single sequencing run permits 42 or 865 surrogate samples and 8 or 153 whole transcriptome samples to be sequenced at the same time on the MiSeq or NextSeq, respectively, at an average of 250 counts/expressed gene. In combination, this results in low sequencing cost/sample without sacrifice of information or quality.

9) Simple, robust protocol requires only a PCR or qPCR instrument, and access to a sequencer.

Citation Format: Milos Babic, Peter Shepard, Joanne Yeakley, Ruchir Shah, Deepak Mav, Raymond B. Nagle, Bruce Seligmann. Differential expression and mechanistic pathways of prostate cancer identified from FFPE tissue using surrogate or whole transcriptome TempO-Seq targeted gene expression assays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1840.