Dataset Information

Reproducible RNA preparation from sugarcane and citrus for functional genomic applications.

ABSTRACT: High-throughput functional genomic procedures depend on the quality of the RNA used. Copurifying molecules can negatively impact the functionality of some plant RNA preparations employed in these procedures. We present a simplified, rapid, and scalable SDS/phenol-based method that provides the high-quantity and -quality RNA required by the newly emerging biotechnology applications. The method is applied to isolating RNA from tissues of two biotechnologically important crop plants, sugarcane and citrus, which provide a challenge due to the presence of fiber, polysaccharides, or secondary metabolites. The RNA isolated by this method is suitable for several downstream applications including northern blot hybridization, microarray analysis, and quantitative RT-PCR. This method has been used in a diverse range of projects ranging from screening plant lines overexpressing mammalian genes to analyzing plant responses to viral infection and defense signaling molecules.

SUBMITTER: Damaj MB

PROVIDER: S-EPMC2817868 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reproducible RNA preparation from sugarcane and citrus for functional genomic applications.

Damaj Mona B MB Beremand Phillip D PD Buenrostro-Nava Marco T MT Riedel Beth B Molina Joe J JJ Kumpatla Siva P SP Thomas Terry L TL Mirkov T Erik TE

International journal of plant genomics 20090101

High-throughput functional genomic procedures depend on the quality of the RNA used. Copurifying molecules can negatively impact the functionality of some plant RNA preparations employed in these procedures. We present a simplified, rapid, and scalable SDS/phenol-based method that provides the high-quantity and -quality RNA required by the newly emerging biotechnology applications. The method is applied to isolating RNA from tissues of two biotechnologically important crop plants, sugarcane and ...[more]

PMID: 20148085

Similar Datasets

Project description:Background:Several high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications. Results:We developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection. Conclusion:To facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation.

Project description:Cell therapy based on dendritic cells (DCs) pulsed with tumor lysate is a promising approach in addition to conventional therapy for the treatment of patients with glioblastoma (GB). The success of this approach strongly depends on the ability to generate high-quality, functionally mature DCs (mDCs), with a high level of standardization and in compliance with Good Manufacturing Practices. In the cell factory of the Carlo Besta Foundation, two phase I clinical trials on immunotherapy with tumor lysate-loaded DCs as treatment for GB are ongoing. From 2010 to 2014, 54 patients were enrolled in the studies and 54 batches of DCs were prepared. We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating yield of mDCs and their quality in terms of microbiological safety and immunological efficacy. The number of mDCs obtained allowed the treatment of all the enrolled patients. All 54 batches were sterile, conformed to acceptable endotoxin levels, and were free of Mycoplasma species and adventitious viruses. During culture, cells maintained a high percentage of viability (87%-98%), and all batches showed high viability after thawing (mean±SD: 94.6%±2.9%). Phenotype evaluation of mDCs showed an evident upregulation of markers typical of DC maturation; mixed lymphocyte reaction tests for the functional evaluation of DCs demonstrated that all batches were able to induce lymphocyte responses. These results demonstrated that our protocol for DC preparation is highly reproducible and permits generation of large numbers of safe and functional DCs for in vivo use in immunotherapy approaches.Cell therapy based on antigen-pulsed dendritic cells (DCs) is a promising approach for the treatment of glioblastoma patients. The success of this approach strongly depends on the ability to generate high-quality, functional DCs with a high level of standardization, ensuring reproducibility, efficacy, and safety of the final product. This article summarizes the results of the quality controls on 54 batches, to demonstrate the feasibility of producing a therapeutic cell-based vaccine via a well-controlled Good Manufacturing Practices (GMP)-compliant production process. The findings may be of scientific interest to those working in the field of preparation of GMP-compliant products for cell-therapy applications.

Project description:BACKGROUND: There had been many reports on genetic transformation of Citrus for functional genomic studies but few included genes associated with flower or fruit traits. A major reason for this might derive from the extensive juvenile stage of Citrus plants when regenerated from juvenile explants (epicotyls, cotyledon or calli), which delays the observation of the resulting phenotype. Alternatives include the use of explants from adult tissues, which sometimes may be recalcitrant to regeneration or transformation, or of early-flowering genotypes. However, there is no report about the use of early-flowering sweet orange mutants for functional genomic studies. RESULTS: Here, we propose a sweet orange spontaneous early-flowering mutant, named 'x11', as a platform for Citrus functional genomic studies, particularly for genes associated with flower or fruit traits. We report a procedure for efficient regeneration and transformation using epicotyl segment explants of 'x11' and Agrobacterium tumefaciens as a proof-of-concept. The average transformation efficiency was 18.6%, but reached 29.6% in the best protocol tested. Among 270 positive shoots, five were in vitro micrografted and acclimatized, followed by evaluation of transgene expression by quantitative amplification of reversed transcripts (RT-qPCR) and determination of the number of copies inserted. Four of these plants, containing from one to four copies of the transgene, exhibited the first flowers within three months after ex vitro establishment, and the other, two months later, regardless of the period of the year. Flowers of transgenic plants displayed fertile pollen and gynoecium, with self-pollination inducing fruit development with seeds. Histochemical staining for ?-glucuronidase activity using stem segments, flowers and fruits from 5 to 7 month-old acclimatized transgenic plants confirmed the constitutive transgene expression in these organs. CONCLUSION: The 'x11' sweet orange is suitable for functional genomics studies with a satisfactory transformation rate, and it can be considered a good model for functional genomic studies in commercial sweet oranges, for traits related to flower and fruit.

Dataset Information

Reproducible RNA preparation from sugarcane and citrus for functional genomic applications.

Publications

Reproducible RNA preparation from sugarcane and citrus for functional genomic applications.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets