Dataset Information

Isolation of high-quality RNA from recalcitrant leaves of variegated and resurrection plants.

ABSTRACT: Resurrection plant Ramonda serbica is a suitable model to investigate mechanisms of desiccation tolerance, while variegated Pelargonium zonale has been proven to serve as an excellent model for the metabolite allocation between sink tissue and source tissue within the same organ. However, the genomes of these plants are still not sequenced, limiting their application in molecular studies. To investigate the transcript abundance by next-generation sequencing, high-quality RNA input is required. Leaves of both P. zonale and R. serbica are rich in polyphenols that interfere with high-quality RNA extraction by common protocols. Moreover, low water content and high amount of sugars and other osmoprotectants in desiccated R. serbica leaves present the additional challenge in total RNA extraction. Here, we evaluated and compared several already established TRIzol- and CTAB-based protocols aiming to develop the efficient, simple and low-cost methods for the extraction of the satisfactory yield RNA of great purity and integrity, required for the construction of high-quality cDNA libraries. Our results show that the CTAB-based protocol (i.e. CTAB 1b) enabled the extraction of high-quality RNA from photosynthetically active and non-photosynthetically active leaf sectors of P. zonale, with high RIN values. On the other hand, TRIzol-based protocol provided a high RNA yield with low contamination and high RNA integrity even in desiccated leaves of R. serbica. We envisage that the proposed protocol would be suitable for the RNA extractions from other desiccated organs (e.g. seeds, grains, pollen grains).

SUBMITTER: Vidovic M

PROVIDER: S-EPMC7266862 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Laser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti.Total RNA was isolated from Ae. aegypti midguts that were either fresh-frozen or fixed with histological fixatives. Generally, fresh-frozen tissue sections are a common source of quality LMM-derived RNA; however, our aim was to develop an LMM protocol that could inactivate pathogenic viruses by fixation, while simultaneously preserving RNA from arbovirus-infected mosquitoes. Three groups (10 - 15 mosquitoes per group) of female Ae. aegypti at 24 or 48-hours post-blood meal were intrathoracically injected with one of seven common fixatives (Bouin's, Carnoy's, Formoy's, Cal-Rite, 4% formalin, 10% neutral buffered formalin, or zinc formalin) to evaluate their effect on RNA quality. Total RNA was isolated from the fixed abdomens using a Trizol® method. The results indicated that RNA from Carnoy's and Bouin's fixative samples was comparable to that of fresh frozen midguts (control) in duplicate experiments. When Carnoy's and Bouin's were used to fix the midguts for the LMM procedure, however, Carnoy's-fixed RNA clearly showed much less degradation than Bouin's-fixed RNA. In addition, a sample of 5 randomly chosen transcripts were amplified more efficiently using the Carnoy's treated LMM RNA than Bouin's-fixed RNA in quantitative real-time PCR (qRT-PCR) assays, suggesting there were more intact target mRNAs in the Carnoy's fixed RNA. The yields of total RNA ranged from 0.3 to 19.0 ng per ~3.0 × 10(6) ?m2 in the LMM procedure.Carnoy's fixative was found to be highly compatible with LMM, producing high quality RNA from Ae. aegypti midguts while inactivating viral pathogens. Our findings suggest that LMM in conjunction with Carnoy's fixation can be applied to studies in Ae. aegypti infected with arboviruses without compromising biosafety and RNA quality. This LMM method should be applicable to other mosquito vector studies.

Dataset Information

Isolation of high-quality RNA from recalcitrant leaves of variegated and resurrection plants.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets