Abstract
Background: Cyst nematodes are biotrophs that form specialized feeding structures in the roots of host plants, which consist of a syncytial fusion of hypertrophied cells. The formation of syncytium is accompanied by profound transcriptional changes and active metabolism in infected tissues. The challenge in gene expression studies for syncytium has always been the isolation of pure syncytial material and subsequent extraction of intact RNA. Root fragments containing syncytium had been used for microarray analyses. However, the inclusion of neighbouring cells dilutes the syncytium-specific mRNA population. Micro-sectioning coupled with laser capture microdissection (LCM) offers an opportunity for the isolation of feeding sites from heterogeneous cell populations. But recovery of intact RNA from syncytium dissected by LCM is complicated due to extended steps of fixation, tissue preparation, embedding and sectioning. Results: In the present study, we have optimized the procedure of sample preparation for LCM to isolate high quality of RNA from cyst nematode induced syncytia in Arabidopsis roots which can be used for transcriptomic studies. We investigated the effect of various sucrose concentrations as cryoprotectant on RNA quality and morphology of syncytial sections. We also compared various types of microscopic slides for strong adherence of sections while removing embedding material. Conclusion: The use of optimal sucrose concentrations as cryoprotection plays a key role in RNA stability and morphology of sections. Treatment with higher sucrose concentrations minimizes the risk of RNA degradation, whereas longer incubation times help maintaining the morphology of tissue sections. Our method allows isolating high-quality RNA from nematode feeding sites that is suitable for downstream applications such as microarray experiments.
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Claims 5
Claim · model-written summary
Optimized preparation yielded high-quality RNA from cyst nematode syncytia for transcriptomic studies.
- Taxon
- Arabidopsis
Arabidopsise0 - Organ / stage
- rootse0
- Interactor
- cyst nematodee0pest
- Outcome
- high quality of RNAe0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“optimized the procedure of sample preparation for LCM to isolate high quality of RNA from cyst nematode induced syncytia in Arabidopsis roots which can be used for transcriptomic studies”
Claim · model-written summary
Optimal sucrose cryoprotection supported RNA stability and section morphology.
- Taxon
- ArabidopsisArabidopsise1
- Organ / stage
- rootse1
- Treatment
- optimal sucrose concentrations as cryoprotectione0
- Outcome
- RNA stability and morphology of sectionse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The use of optimal sucrose concentrations as cryoprotection plays a key role in RNA stability and morphology of sections”
e1“Arabidopsis roots”
Claim · model-written summary
Higher sucrose concentrations reduced RNA degradation risk.
- Taxon
- ArabidopsisArabidopsise1
- Treatment
- higher sucrose concentrationse0
- Outcome
- risk of RNA degradatione0
- Direction
- minimizese0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Treatment with higher sucrose concentrations minimizes the risk of RNA degradation”
e1“Arabidopsis roots”
Claim · model-written summary
Longer incubation maintained tissue section morphology.
- Taxon
- ArabidopsisArabidopsise1
- Treatment
- longer incubation timese0
- Outcome
- morphology of tissue sectionse0
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“longer incubation times help maintaining the morphology of tissue sections”
e1“Arabidopsis roots”
Claim · model-written summary
The method recovered high-quality RNA suitable for microarray experiments from nematode feeding sites.
- Taxon
- ArabidopsisArabidopsise1
- Organ / stage
- rootse1
- Outcome
- high-quality RNAe0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Our method allows isolating high-quality RNA from nematode feeding sites that is suitable for downstream applications such as microarray experiments”
e1“Arabidopsis roots”
The same questions in other papers
sucrose · growth · ArabidopsisPositive and negative results
8 papers in the same direction as this paper · 8 in the opposite direction
- negative
- negative
- negative
13 papers more in this group
See all 17 papers →Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.