scholarly journals Quantifying BRCA1 and BRCA2 mRNA Isoform Expression Levels in Single Cells

2019 ◽  
Vol 20 (3) ◽  
pp. 693 ◽  
Author(s):  
Vanessa Lattimore ◽  
John Pearson ◽  
Arthur Morley-Bunker ◽  
kConFab Investigators ◽  
Amanda Spurdle ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Single Cells ◽  
In Situ Hybridisation ◽  
Brca1 And Brca2 ◽  
Mrna Isoforms ◽  
Absolute Expression ◽  
Mrna Isoform ◽  
Splicing Patterns ◽  
Diagnostic Setting

BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 ∆17_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting.

scholarly journals mRNA detection in budding yeast with single fluorophores

2017 ◽  
Author(s):  
Gable M. Wadsworth ◽  
Rasesh Y. Parikh ◽  
John S. Choy ◽  
Harold D. Kim
Keyword(s):  
Single Molecule ◽  
Budding Yeast ◽  
Single Cells ◽  
Phenotypic Variability ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mrna Isoform

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect more than 50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

scholarly journals Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

2020 ◽  
Vol 5 ◽  
pp. 76
Author(s):  
John C. W. Hildyard ◽  
Abbe H. Crawford ◽  
Faye Rawson ◽  
Dominique O. Riddell ◽  
Rachel C. M. Harron ◽  
...  
Keyword(s):  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Dystrophin Gene ◽  
Tissue Expression ◽  
Mrna Isoforms ◽  
Mammalian Embryo ◽  
Isoform Expression ◽  
Single Transcript ◽  
The Brain

Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.

scholarly journals Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

2020 ◽  
Vol 5 ◽  
pp. 76
Author(s):  
John C. W. Hildyard ◽  
Abbe H. Crawford ◽  
Faye Rawson ◽  
Dominique O. Riddell ◽  
Rachel C. M. Harron ◽  
...  
Keyword(s):  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Dystrophin Gene ◽  
Tissue Expression ◽  
Mrna Isoforms ◽  
Mammalian Embryo ◽  
Isoform Expression ◽  
Single Transcript ◽  
The Brain

Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.

Nuclear-specific accumulation of telomerase reverse transcriptase (TERT) mRNA in TERT promoter mutated follicular thyroid tumours visualised by in situ hybridisation: a possible clinical screening tool?

2021 ◽  
pp. jclinpath-2021-207631
Author(s):  
L Samuel Hellgren ◽  
Ann Olsson ◽  
Ann Kaufeldt ◽  
Johan O Paulsson ◽  
Martin Hysek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reverse Transcriptase ◽  
Mrna Expression ◽  
In Situ Hybridisation ◽  
Telomerase Reverse Transcriptase ◽  
Mrna Levels ◽  
Specific Accumulation ◽  
Or Gene ◽  
Promoter Mutations

AimsUpregulation of the telomerase reverse transcriptase (TERT) gene is a frequent finding in follicular thyroid carcinomas (FTCs) with metastatic features. The augmented expression is usually caused by TERT promoter mutations. As TERT protein immunohistochemistry might not correlate to TERT mRNA levels in follicular thyroid tumours, we therefore sought to determine if visualisation of TERT mRNA through in situ hybridisation could highlight high-risk cases.MethodsWe collected formalin-fixated paraffin-embedded tissues from 26 follicular thyroid tumours; 7 FTCs, 2 follicular thyroid tumours of uncertain malignant potential (FT-UMPs) and a single Hürthle cell carcinoma with established TERT promoter mutations and gene expression, as well as 16 FTCs with no TERT gene aberrancy or gene expression, and assessed them using RNA Scope in situ hybridisation (ISH) and TERT probes targeting the two main TERT transcripts (TERT1 and TERT2).ResultsTERT 1 and/or 2 mRNA was found by ISH in 8/10 cases with established promoter mutations and mRNA expression, whereas all 16 cases without TERT gene aberrancies or gene expression were negative (Fisher’s exact p<0.001). Strikingly, TERT mRNA was visualised in the nuclear compartment only, thereby corroborating earlier studies suggesting a non-conventional role for TERT in tumour biology. Moreover, TERT mRNA expression was scattered across the tissue sections and only found in a few percentages of tumour nuclei.ConclusionsTERT mRNA seems to be focally expressed and localised exclusively to the nucleus in TERT promoter mutated follicular thyroid tumours, possibly reflecting a true biological and unorthodox phenomenon worthy of further investigations.

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