Enriched transcriptome analysis of laser capture microdissected populations of single cells to investigate intracellular heterogeneity in immunostained FFPE sections

Hammoudeh, Sarah; Hammoudeh, Arabella Musa; Venkatachalam, Thenmozhi; Rawat, Surendra; Jayakumar, Manju Nidagodu; Rahmani, Mohamed; Hamoudi, Rifat

doi:10.1016/j.csbj.2021.09.010

Cited by 5 publications

(7 citation statements)

References 61 publications

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“…Whole transcriptome library was prepared from both unamplified and amplified RNA using the Ion AmpliSeq human gene expression on S5 system (Thermo Fisher Scientific, USA) as previously described [ 51 ]. For each FFPE sample, ∼10 ng of gDNA-free RNA was used to prepare barcoded libraries using Ion AmpliSeq transcriptome human gene expression kit (Thermo Fisher Scientific, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The libraries were diluted to 100 pM, pooled together, amplified using emulsion PCR on the Ion One Touch 2 (OT2) instrument, and enriched using the Ion One Touch ES as per manufacturer's instructions. RNA-sequencing of the libraries was performed using Ion S5 XL Semiconductor sequencer on Ion 540 Chip (Thermo Fisher Scientific, USA) as previously described [ 51 ].…”

Section: Methodsmentioning

confidence: 99%

“…Ion AmpliSeq Human gene expression panel used in library preparation amplifies targeted regions of approximately 21,000 genes. Base called and aligned sequences were normalized using Fragments per kilobase million (FPKM) normalization according to Hammoudeh et al [ 51 ]. In general, libraries prepared from FFPE samples result in large amounts of short fragment sequences.…”

Section: Methodsmentioning

confidence: 99%

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LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

Bhamidimarri,

Salameh,

Mahdami

et al. 2024

Heliyon

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

Bhamidimarri,

Salameh,

Mahdami

et al. 2024

Heliyon

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“…Several institutions, including the Mayo Clinic, currently employ LCM to enrich for amyloidogenic proteins before further analysis with mass spectrometry on the polypeptides [69, 70]. While LCM remains popular in basic science research, forays into clinical practice, such as digital droplet PCR and RNA-seq for clinically relevant gene expression tests, are on the horizon [71, 72].…”

Section: Microdissection Methodsmentioning

confidence: 99%

A Comparison of Tissue Dissection Techniques for Diagnostic, Prognostic, and Theragnostic Analysis of Human Disease

Walsh

Halushka

2022

Pathobiology

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Histopathology has historically been the critical technique for the diagnosis and treatment of human disease. Today, genomics, transcriptomics, and proteomics from specific cells, rather than bulk tissue, have become key to understanding underlying disease mechanisms and rendering useful diagnostic information. Extraction of desired analytes, i.e., nucleic acids or proteins, from easily accessible formalin-fixed paraffin-embedded tissues allows for clinically relevant activities, such as sequencing biomarker mutations or typing amyloidogenic proteins. Genetic profiling has become routine for cancers as varied as non-small cell lung cancer and prostatic carcinoma. The five main tissue dissection techniques that have been developed thus far include: bulk scraping, manual macrodissection, manual microdissection, laser-capture microdissection, and expression microdissection. In this review, we discuss the importance of tissue dissection in clinical practice and research, the basic methods, applications, as well as some advantages and disadvantages for each modality.

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“…Next, the constructed library was purified using Agencourt AMPure XP Beads (Beckman Coulter, Cat # A63881, Indianapolis, USA), quantified using an Ion Library TaqMan™ Quantitation Kit (Applied Biosystems, Cat # 4468802, Waltham, USA), further diluted to 100 pM, pooled equally, and amplified using emulsion PCR on Ion OneTouch™ 2 instrument (OT2) (Thermo Fisher, RRID:SCR_023289, Cat # 4474778, USA) and the enrichment was performed on Ion OneTouch™ ES (Thermo Fisher, Cat # 4469495, RRID:SCR_023290, USA). 18 Finally, RNA-sequencing was performed on the Ion S5 XL Semiconductor sequencer using the Ion 540-Chip (Life Technologies, Cat # A27765, California, USA).…”

Section: Whole Transcriptome Sequencingmentioning

confidence: 99%

Impact of estrogen and progesterone hormone receptors on the progression of interferon-γ sensitized breast cancer cells

Shihab,

Bouzid,

Yener

et al. 2023

F1000Res

Self Cite

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Background: Breast cancer is a chronic complex disease. Its progression depends partly on the interaction between tumor and immune cells. Whilst immunotherapy is the new promising treatment, many patients with breast cancer acquire resistance. Interferon gamma (IFN-γ) is a pleiotropic cytokine that is primarily released by T cells and natural killer (NK) cells and has always been praised for its antitumor activities. However, IFN-γ may induce different modulations in breast cancer cells that are expressing or not expressing the hormone receptors estrogen and progesterone. Methods: In this study, to examine the effect of IFN-γ on the subtypes of breast cancer in relation to the expression of estrogen and progesterone genes, we performed RNA-sequencing on the triple negative cells MDA-MB231 and ER/PR transfected MDA-MB231 cells (untreated or treated with 100 ng/ml IFN-γ). Various bioinformatics analyses were performed to investigate the affected functional pathways, and immune genes related to the different types of breast cancer cells. Results: The set of differentially expressed genes (DEGs) that are regulated by IFN-γ were unique, and specific to each breast cancer subtype. These unique DEG patterns in hormone-positive cells (GBP3, HLA-DPA1, HLA-DRB1, HLA-E, IL6) and triple negative cells (IFI6, ISG15, CCL5) showed significant but distinct effects on patients’ overall survival as well as noticeable differences in immune modulation and regulation. Conclusions: IFN-γ signaling can differentially affect the pattern of gene expression in breast cancer cells in an estrogen receptor (ER) / progesterone receptor (PR)-dependent manner. IFN-γ treatment of ER+/PR+ breast cancer cells upregulated the expression of genes related to immune cells and showed improved patient prognosis, while TNBC showed negative regulation of the expression of genes related to immune cells and worse patient prognosis.

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Enriched transcriptome analysis of laser capture microdissected populations of single cells to investigate intracellular heterogeneity in immunostained FFPE sections

Abstract: Graphical abstract

Cited by 5 publications

References 61 publications

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

A Comparison of Tissue Dissection Techniques for Diagnostic, Prognostic, and Theragnostic Analysis of Human Disease

Impact of estrogen and progesterone hormone receptors on the progression of interferon-γ sensitized breast cancer cells

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