Hexadecyltrimethylammonium Bromide (CTAB)-based Protocol to Isolate High-Quality RNA in Adequate Quantities for Gene Expression Analyses in Developing Seeds of Lentils (Lens culinaris Medik.)

Abstract Fireflies are among the most charismatic insects for their spectacular bioluminescence, but the origin and evolution of bioluminescence remain elusive. Especially, the genic basis of luciferin (d-luciferin) biosynthesis and light patterns is largely unknown. Here, we present the high-quality reference genomes of two fireflies Lamprigera yunnana (1053 Mb) and Abscondita terminalis (501 Mb) with great differences in both morphology and luminous behavior. We sequenced the transcriptomes and proteomes of luminous organs of two species. We created the CRISPR/Cas9-induced mutants of Abdominal B gene without luminous organs in the larvae of A. terminalis and sequenced the transcriptomes of mutants and wild-types. Combining gene expression analyses with comparative genomics, we propose a more complete luciferin synthesis pathway, and confirm the convergent evolution of bioluminescence in insects. Using experiments, the function of the firefly acyl-CoA thioesterase (ACOT1) to convert l-luciferin to d-luciferin was validated for the first time. Comparisons of three-dimension reconstruction of luminous organs and their differentially expressed genes among two species suggest that two positive genes in the calcium signaling pathway and structural difference of luminous organs may play an important role in the evolution of flash pattern. Altogether, our results provide important resources for further exploring bioluminescence in insects.

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Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels

Open Biology ◽

10.1098/rsob.200388 ◽

2021 ◽

Vol 11 (6) ◽

pp. 200388

Author(s):

Anna Jaeschke ◽

Nicholas R. Harvey ◽

Mikhail Tsurkan ◽

Carsten Werner ◽

Lyn R. Griffiths ◽

...

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Three Dimensional ◽

3D Cell Culture ◽

Biomechanical Properties ◽

High Quality ◽

Gene Expression Analyses

Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro . Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.

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Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses

Plant Omics ◽

10.21475/poj.10.04.17.pne745 ◽

2017 ◽

Vol 10 (04) ◽

pp. 205-209

Author(s):

Edgardo H. Avico ◽

◽

Raúl M. Acevedo ◽

Pablo I. Calzadilla ◽

Oscar A. Ruiz ◽

...

Keyword(s):

Gene Expression ◽

Next Generation Sequencing ◽

Ilex Paraguariensis ◽

Next Generation ◽

High Quality ◽

Gene Expression Analyses ◽

Generation Sequencing

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Big data from small tissues: extraction of high-quality RNA for different plant tissue types during oilseed Brassica spp. seed development for RNA-Sequencing

10.1101/2019.12.20.885012 ◽

2019 ◽

Author(s):

Laura Siles ◽

Peter Eastmond ◽

Smita Kurup

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Seed Development ◽

Developmental Stages ◽

Rna Extraction ◽

Rna Isolation ◽

Extraction Methods ◽

High Quality ◽

Gene Expression Analyses ◽

Rna Extraction Methods

AbstractObtaining high-quality RNA for gene expression analyses from different seed tissues is challenging due to the presence of various contaminants, such as polyphenols, polysaccharides and lipids which interfere with RNA extraction methods. At present, the available protocols for extracting RNA from seeds require high amounts of tissue and are mainly focused on extracting RNA from whole seeds. However, extracting RNA at the tissue level enables more detailed studies regarding tissue specific transcriptome during development. Seeds from heart stage embryo to mature developmental stages of Brassica napus and B. oleracea were sampled for isolation of the embryo, endosperm and seed coat tissues. Ovules and gynoecia wall tissue were also collected at the pre-fertilization stage. After testing several RNA extraction methods, E.Z.N.A. Plant RNA Kit and Picopure RNA Isolation kit extraction methods with some modifications, as well as the use of PVPP for seed coats and endosperms at green stages, resulted in high RNA concentrations with clear 28S and 18S bands and high RIN values. Here, we present efficient and reliable RNA extraction methods for different genotypes of Brassica spp for different tissue types during seed development. The high-quality RNA obtained by using these methodologies is suitable for RNA-Sequencing and gene expression analyses.

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