Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

Arts, Peer; Raadt, Jori van der; Gestel, Sebastianus H. C. van; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

doi:10.1038/ncomms15190

Cited by 16 publications

(18 citation statements)

References 32 publications

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“…RNA was isolated from 10 μm cryosections using TRIzol reagent (ThermoFisher Scientific, Waltham, MA, USA) and reverse transcribed with Superscript II (ThermoFisher Scientific) using random hexamer primers, according to the manufacturer's instructions. Targeted RNA sequencing using smMIPs has been described before (16, 22). In short, smMIPs were designed against target regions of interest (UCSC human genome assembly hg19 and splice-variant specific FASTA sequences) based on the MIPgen algorithm as described by Boyle et al (23), including a random octanucleotide unique molecule identifier (UMI).…”

Section: Methodsmentioning

confidence: 99%

Molecular Profiling of Druggable Targets in Clear Cell Renal Cell Carcinoma Through Targeted RNA Sequencing

et al. 2019

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Clear cell renal cell carcinoma (ccRCC) comprises more than 80% of all renal cancers and when metastasized leads to a 5-year survival rate of only 10%. The high rate of therapy failure and resistance development calls for reliable methods that provide information on the actionable biological pathways and predict optimal treatment protocols for individual patients. We here applied targeted RNA sequencing (t/RNA-NGS) using single molecule Molecular Inversion Probes on tumor nephrectomy samples of five ccRCC patients, comparing tumor with healthy kidney tissues. Transcriptome profiling focused on expression of genes with involvement in ccRCC biology that can be targeted with clinically available drugs. Results confirm high expression of vascular endothelial growth factor-A (VEGF-A) in tumor tissue relative to healthy-appearing kidney, in line with the angiogenic nature of ccRCC. PDGFRα and KIT, targets of the multi-kinase inhibitor sunitinib which is one of the current choices of first-line drug in metastasized ccRCC patients, were expressed at relatively low levels in tumor tissues, whereas significantly increased in normal kidney. Of all measured druggable tyrosine kinases, MET, AXL, or EGFR were expressed at higher levels in tumors than in normal kidney tissues, although intertumor differences were observed. Using cancer cell lines we show that t/RNA-NGS gene expression profiles can be used to predict in vitro sensitivity to targeted drugs. In conclusion, t/RNA-NGS analysis may provide insights into the (druggable) molecular make-up of individual renal cancers, and may guide personalized therapy of renal cell cancers.

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Section: Methodsmentioning

confidence: 99%

Molecular Profiling of Druggable Targets in Clear Cell Renal Cell Carcinoma Through Targeted RNA Sequencing

et al. 2019

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“…RNA was isolated from 14 cell lines using TRIzol reagent (ThermoFisher Scientific, Waltham, MA, USA) and reverse transcribed with Superscript II (ThermoFisher Scientific) using random hexamer primers, according to the manufacturer's instructions. Targeted RNA sequencing using smMIPs has been described in detail before [16,29].…”

Section: Single Molecule Molecular Inversion Probe Sequencingmentioning

confidence: 99%

ACLY (ATP Citrate Lyase) Mediates Radioresistance in Head and Neck Squamous Cell Carcinomas and is a Novel Predictive Radiotherapy Biomarker

Göttgens

Heuvel

Jong

et al. 2019

Cancers

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Radiotherapy is an important treatment modality of head and neck squamous cell carcinomas (HNSCC). Multiple links have been described between the metabolic activity of tumors and their clinical outcome. Here we test the hypothesis that metabolic features determine radiosensitivity, explaining the relationship between metabolism and clinical outcome. Radiosensitivity of 14 human HNSCC cell lines was determined using colony forming assays and the expression profile of approximately 200 metabolic and cancer-related genes was generated using targeted RNA sequencing by single molecule molecular inversion probes. Results: Correlation between radiosensitivity data and expression profiles yielded 18 genes associated with radiosensitivity or radioresistance, of which adenosine triphosphate (ATP) citrate lyase (ACLY) was of particular interest. Pharmacological inhibition of ACLY caused an impairment of DNA damage repair, specifically homologous recombination, and lead to radiosensitization in HNSCC cell lines. Examination of a The Cancer Genome Atlas (TCGA) cohort of HNSCC patients revealed that high expression of ACLY was predictive for radiotherapy failure, as it was only associated with poor overall survival in patients who received radiotherapy (hazard ratio of 2.00, 95% CI: 1.12–3.55; p = 0.0184). These data were further validated in an independent cohort of HNSCC patients treated with chemoradiation. Furthermore, patients with poor locoregional control after radiotherapy have significantly higher nuclear ACLY protein levels. Together, we here show that ACLY affects DNA damage repair, and is a predictive factor for radiotherapy outcome in HNSCC.

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“…RNA from cell lines and snap-frozen xenograft tissue was isolated and converted to cDNA as described under ‘PCR’. The smMIP method and its application for targeted transcriptome sequencing has been described before 20 , 21 and is depicted in Fig. 1a .…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we combine single molecule molecular inversion probe (smMIP) targeted transcriptome sequencing 20 , 21 (Fig. 1 ) with in situ detection of transcript splice variants using padlock probe-based rolling circle amplification 22 – 26 (Fig.…”

Section: Introductionmentioning

confidence: 99%

Quantification and localization of oncogenic receptor tyrosine kinase variant transcripts using molecular inversion probes

Heuvel

Das

Bitter

et al. 2018

Sci Rep

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Oncogenic membrane receptor tyrosine kinases such as MET and EGFR, or auto-active variants thereof, are important targets for cancer precision therapy. Targeted inhibition of these oncogenic receptors however invariably leads to resistance, resulting from acquisition of resistance-inducing mutations or from selective outgrowth of a priori resistant tumour cells. Most applied molecular protocols cannot distinguish between intracellular and intercellular heterogeneity of oncogene (variant) expression, which may lead to misinterpretation of the molecular make-up of a cancer and suboptimal application of targeted therapies. We here combined two related techniques to allow semiquantitative and localized in situ detection of specific transcript splice variants using single molecule molecular inversion probe (smMIP)-based next generation sequencing and padlock probe-based rolling circle amplification, respectively. We show highly specific padlock probe-based multiplex detection of MET, METΔ7-8 and METΔ14 transcripts, lacking exons 7–8 and exon 14 respectively, and of EGFR and the auto-active EGFRvIII, lacking exons 2–7. The combination of quantitative transcript variant detection with smMIPs and transcript localization using padlock probes can be used for detection of oncogenic transcripts on the single-cell level, allowing study of tumour heterogeneity. Visualization of tumour heterogeneity can shed light on the biology underlying drug resistance and potentially improve targeted therapeutics.

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Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

Cited by 16 publications

References 32 publications

Molecular Profiling of Druggable Targets in Clear Cell Renal Cell Carcinoma Through Targeted RNA Sequencing

Molecular Profiling of Druggable Targets in Clear Cell Renal Cell Carcinoma Through Targeted RNA Sequencing

ACLY (ATP Citrate Lyase) Mediates Radioresistance in Head and Neck Squamous Cell Carcinomas and is a Novel Predictive Radiotherapy Biomarker

Quantification and localization of oncogenic receptor tyrosine kinase variant transcripts using molecular inversion probes

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