A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

AbstractBackgroundRNAs to be used in transcriptome analysis must be of high quality and pure in order to ensure maximum representation of the expressed genes. RNA isolation is difficult in hazelnut tissues containing large amounts of secondary metabolite, phenolic compounds and the cell wall structure. Commonly used protocols for RNA isolation are those that require a lot of labor and time and also do not allow sufficient RNA isolation when applied to tissues rich in phenolic compounds. This study was aimed to develop an efficient method for isolation of total RNAs from bud of hazelnut to be used in RNA sequencing.Materials and methodsAn optimized new method was successfully applied on three different hazelnuts genotypes (Çakıldak, Palaz, Tombul) and about 25 times higher amount of total RNAs per mg fresh tissues were obtained compared to classical CTAB method. Different methods have been tried for the isolation of RNA from hazelnut tissues and the determination of the quality of the obtained RNAs.ResultsThe quality and quantity of isolalated total RNAs were determined by spectrophotometer, electrophoresis and PCR. This success has been caught without any compromise of purity since A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were >2.0 in all purified RNAs.ConclusionThe total RNAs isolated with new protocol was found to be suitable for RNA sequencing and other molecular applications.

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High-quality total RNA isolation from melon (Cucumis melo L.) fruits rich in polysaccharides

Semina Ciências Agrárias ◽

10.5433/1679-0359.2017v38n4p2201 ◽

2017 ◽

Vol 38 (4) ◽

pp. 2201 ◽

Cited By ~ 1

Author(s):

Gabrielle Silveira de Campos ◽

Ricardo Antônio Ayub ◽

Rafael Mazer Etto ◽

Carolina Weigert Galvão ◽

Marília Aparecida Stroka ◽

...

Keyword(s):

Rna Extraction ◽

Sodium Perchlorate ◽

Rna Isolation ◽

World Market ◽

Molecular Techniques ◽

Guanidine Thiocyanate ◽

High Quality ◽

Total Rna ◽

High Concentrations ◽

Cucumis Melo L

Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazil’s main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1) guanidine thiocyanate/phenol/chloroform; T2) sodium azide/?-mercaptoethanol; T3) phenol/guanidine thiocyanate; T4) CTAB/PVP/?-mercaptoethanol; T5) SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6) sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.

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Extensive phylogenies of human development reveal variable embryonic patterns

10.1101/2020.11.25.397828 ◽

2020 ◽

Author(s):

Tim H. H. Coorens ◽

Luiza Moore ◽

Philip S. Robinson ◽

Rashesh Sanghvi ◽

Joseph Christopher ◽

...

Keyword(s):

Progenitor Cell ◽

Spatial Effect ◽

Lineage Tracing ◽

Substantial Variation ◽

Normal Tissues ◽

Multiple Organs ◽

Naturally Occurring ◽

Adult Body ◽

Laser Capture ◽

Laser Capture Microdissected

ABSTRACTStarting from the zygote, all cells in the developing and adult human body continuously acquire mutations. A mutation shared between two different cells implies a shared progenitor cell and can thus be used as a naturally occurring marker for lineage tracing. Here, we reconstruct extensive phylogenies of normal tissues from three adult individuals using whole-genome sequencing of 511 laser capture microdissected samples from multiple organs. Early embryonic progenitor cells inferred from the phylogenies often contribute in different proportions to the adult body and the extent of this asymmetry is variable between individuals, with ratios between the first two reconstructed cells ranging from 56:44 to 92:8. Asymmetries also pervade subsequent cell generations and can differ between tissues in the same individual. The phylogenies also resolve the spatial embryonic origins and patterning of tissues, revealing a spatial effect in the development of the human brain. Supplemented by data on eleven men, we timed the split between soma and germline, with the earliest observed segregation occurring at the first cell divisions. This research demonstrates that, despite reaching the same ultimate tissue patterns, early bottlenecks and lineage commitments lead to substantial variation in embryonic patterns both within and between individuals.

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Differential stromal reprogramming in benign and malignant naturally occurring canine mammary tumours identifies disease-promoting stromal components

10.1101/783621 ◽

2019 ◽

Author(s):

Parisa Amini ◽

Sina Nassiri ◽

Alexandra Malbon ◽

Enni Markkanen

Keyword(s):

Screening Method ◽

Malignant Tumours ◽

Differential Abundance ◽

Naturally Occurring ◽

Mammary Tumours ◽

Ffpe Specimen ◽

Summary Statement ◽

Benign Tumours ◽

Laser Capture ◽

Laser Capture Microdissected

AbstractThe importance of cancer-associated stroma (CAS) for initiation and progression of cancer is well accepted. However, as stromal changes in benign forms of naturally occurring tumours are poorly understood, it remains unclear how CAS from benign and malignant tumours compare. Spontaneous canine mammary tumours are viewed as excellent models of human mammary carcinomas (mCA). We have recently reported highly conserved stromal reprogramming between canine and human mCA based on transcriptome analysis of laser-capture-microdissected FFPE specimen. To identify stromal changes between benign and malignant mammary tumours, we have analysed CAS and matched normal stroma from 13 canine mammary adenomas and compared them to 15 canine mCA. Our analyses revealed distinct stromal reprogramming even in small benign tumours. While similarities in stromal reprogramming exist, the CAS signature clearly distinguished adenomas from mCA, suggesting that it may reliably discriminate between benign and malignant tumours. We identified strongly discriminatory genes and found strong differential enrichment in several hallmark signalling pathways between benign and malignant CAS. The distinction between CAS from adenoma and mCA was further substantiated by differential abundance in cellular composition. Finally, to determine key players in CAS reprograming between adenomas and mCA, a network-based gene screening method identified modules of co-expressing genes with distinct expression profile in benign and malignant CAS, and revealed several hub genes as potential molecular drivers in CAS. Given the relevance of canine CAS as a model for the human disease, our approach identifies potential stromal drivers of tumour malignancy with implications for human mCA.Summary statementRNAsequencing-based analysis of stromal reprogramming between benign and malignant naturally occurring canine mammary tumours identifies potential molecular drivers in cancer-associated stroma that support tumour growth and malignancy.

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