SureSelectXT RNA Direct: A Technique for Expression Analysis Through Sequencing of Target-Enriched FFPE Total RNA

Jones, Jennifer Carter; Siebold, Alex P.; Livi, Carolina B.; Lucas, Anne Bergstrom

doi:10.1007/978-1-4939-7834-2_4

Cited by 4 publications

(5 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…cDNA libraries of formalin-fixed paraffin-embedded blocks cut in 10-μm-thick sections were prepared with the SureSelect XT RNA Direct Reagent Kit (Agilent) [ 32 ]. Briefly, total RNA was isolated from each sample.…”

Section: Methodsmentioning

confidence: 99%

“…cDNA was synthetized from mRNA through reverse transcription [ 33 ]. DV200, defined as the percentage of RNA fragments > 200 nucleotides [ 32 ], was > 50% in most and > 30% in all samples, with no significant difference among the groups ( Table S1 ). Paired-end mRNA was sequenced on the NovaSeq 6000 Sequencing System (RRID:SCR_016387; Illumina, San Diego, CA, USA) using the NovaSeq 6000 S4 Reagent Kit (Illumina).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Non-Muscle-Invasive Bladder Carcinoma with Respect to Basal Versus Luminal Keratin Expression

Jung

Jang

Kim

et al. 2020

IJMS

View full text Add to dashboard Cite

Non-muscle-invasive bladder cancer (NMIBC) consists of transcriptional subtypes that are distinguishable from those of muscle-invasive cancer. We aimed to identify genetic signatures of NMIBC related to basal (K5/6) and luminal (K20) keratin expression. Based on immunohistochemical staining, papillary high-grade NMIBC was classified into K5/6-only (K5/6High-K20Low), K20-only (K5/6Low-K20High), double-high (K5/6High-K20High), and double-low (K5/6Low-K20Low) groups (n = 4 per group). Differentially expressed genes identified between each group using RNA sequencing were subjected to functional enrichment analyses. A public dataset was used for validation. Machine learning algorithms were implemented to predict our samples against UROMOL subtypes. Transcriptional investigation demonstrated that the K20-only group was enriched in the cell cycle, proliferation, and progression gene sets, and this result was also observed in the public dataset. The K5/6-only group was closely regulated by basal-type gene sets and showed activated invasive or adhesive functions. The double-high group was enriched in cell cycle arrest, macromolecule biosynthesis, and FGFR3 signaling. The double-low group moderately expressed genes related to cell cycle and macromolecule biosynthesis. All K20-only group tumors were classified as UROMOL “class 2” by the machine learning algorithms. K5/6 and K20 expression levels indicate the transcriptional subtypes of NMIBC. The K5/6Low-K20High expression is a marker of high-risk NMIBC.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Non-Muscle-Invasive Bladder Carcinoma with Respect to Basal Versus Luminal Keratin Expression

Jung

Jang

Kim

et al. 2020

IJMS

View full text Add to dashboard Cite

show abstract

“…The ribosomal RNA depletion approach has been widely accepted as the preferred method of RNA isolation for degraded or low-input samples 16 – 18 . Another improvement comprising use of an “RNA Direct” workflow including targeted capture using the Agilent Strand-Specific RNA Library Prep kit has also been described 19 .…”

Section: Introductionmentioning

confidence: 99%

Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples

Newton¹,

Sedgewick²,

Cisneros³

et al. 2020

Sci Rep

View full text Add to dashboard Cite

Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

show abstract

“…To analyse gene expression profiles of our samples, we used a targeted whole transcriptome sequencing approach reported as highly accurate and perfectly comparable with RNA-sequencing approaches and next generation sequencing platforms utilized to obtain TCGA data 16 . Although TCGA samples are fresh frozen and we analysed only formalin-fixed and paraffin-embedded (FFPE) tissues, the comparison of transcriptome data is supported by several evidences demonstrating a high correlation of gene expression profiles between these two types of specimens 17–19 . Anyway, in order to overcome this potential limitation, a validation of results was carried out on an independent cohort of PSC, LUAD and LUSC FFPE tissues from our archives.…”

Section: Discussionmentioning

confidence: 94%

Whole transcriptome targeted gene quantification provides new insights on pulmonary sarcomatoid carcinomas

Alì

Bruno

Poma

et al. 2019

Sci Rep

View full text Add to dashboard Cite

Pulmonary sarcomatoid carcinomas (PSC) are a rare group of lung cancer with a median overall survival of 9–12 months. PSC are divided into five histotypes, challenging to diagnose and treat. The identification of PSC biomarkers is warranted, but PSC molecular profile remains to be defined. Herein, a targeted whole transcriptome analysis was performed on 14 PSC samples, evaluated also for the presence of the main oncogene mutations and rearrangements. PSC expression data were compared with transcriptome data of lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC) from The Cancer Genome Atlas. Deregulated genes were used for pathway enrichment analysis; the most representative genes were tested by immunohistochemistry (IHC) in an independent cohort (30 PSC, 31 LUAD, 31 LUSC). All PSC cases were investigated for PD-L1 expression. Thirty-eight genes deregulated in PSC were identified, among these IGJ and SLMAP were confirmed by IHC. Moreover, Forkhead box signaling and Fanconi anemia pathways were specifically enriched in PSC. Finally, some PSC harboured alterations in genes targetable by tyrosine kinase inhibitors, as EGFR and MET . We provide a deep molecular characterization of PSC; the identification of specific molecular profiles, besides increasing our knowledge on PSC biology, might suggest new strategies to improve patients management.

show abstract

SureSelectXT RNA Direct: A Technique for Expression Analysis Through Sequencing of Target-Enriched FFPE Total RNA

Cited by 4 publications

References 4 publications

Non-Muscle-Invasive Bladder Carcinoma with Respect to Basal Versus Luminal Keratin Expression

Non-Muscle-Invasive Bladder Carcinoma with Respect to Basal Versus Luminal Keratin Expression

Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples

Whole transcriptome targeted gene quantification provides new insights on pulmonary sarcomatoid carcinomas

Contact Info

Product

Resources

About