Selective Depletion of Abundant RNAs to Enable Transcriptome Analysis of Low-Input and Highly Degraded Human RNA.

Ribosomal RNAs (rRNAs) are extremely abundant, often constituting 80% to 90% of total RNA. Since rRNA sequences are often not of interest in genomic RNA sequencing experiments, rRNAs can be removed from the sample before the library preparation step, in order to prevent the majority of the library and the majority of sequencing reads from being rRNA. Removal of rRNA can be especially challenging for low quality and formalin-fixed paraffin-embedded (FFPE) RNA samples due to the fragmented nature of these RNA molecules.

Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue.

We report a method for Selective Depletion of abundant RNA (SDRNA) species from Human total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue, here demonstrating removal of ribosomal and mitochondrial transcripts from clinical FFPE tissue RNA archived up to 20 years. Importantly, SDRNA removes 98% of targeted RNAs while preserving relative abundance profiles of non-targeted RNAs, enabling routine whole transcriptome analysis of clinically valuable archival tissue specimens by Next-Generation Sequencing.

Whole transcriptome RNA-Seq analysis of breast cancer recurrence risk using formalin-fixed paraffin-embedded tumor tissue.

RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing.

Mutation detection by real-time PCR: a simple, robust and highly selective method.

Background: Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR.

Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax.

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive.