Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules
Abstract
:1. Introduction
2. Results
2.1. Proteinase K Treatment Time
2.2. Probe Length
2.3. Probe Concentration
2.4. Hybridisation Temperature
2.5. Washing Conditions
2.6. Use of Improved RNA ISH Protocol for Detection of Genes Associated with Seed Development, VvTAU and VvHB63
3. Discussion
4. Materials and Methods
4.1. Plant Materials and Sample Collection
4.2. Preparation of Transcription Template
4.3. In Vitro Transcription Reaction
4.4. Probe Hydrolysis (Optional)
4.5. The In Situ Hybridisation
4.6. Sectioning
4.7. Deproteinisation
4.8. Hybridisation
4.9. Post-Hybridisation
4.10. Detection
5. Conclusions
- (1)
- Pre-hybridisation—proteinase K treatment time of 30 min;
- (2)
- Hybridisation—probe concentration of 100 ng/mL, probe length of 150 bp and temperature of 50 °C;
- (3)
- Post-hybridisation washing—three washes with 0.2× SSC solution and blocking with 1% blocking reagent for 45 min.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Forward Primer Sequence | Reverse Primer Sequence |
---|---|---|
VvTAU | 5′ ATTTTCATGGAGTTTTTCAGCAGAT 3′ | 5′ TGTACAGCATTCACATCTCATCTTA 3′ |
VvHB63 | 5′ TGAACAAGCAGCAGGGAGGAATAG3′ | 5′ ATATTACACATGCTAGGAATTCCAA 3′ |
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Yao, J.; Li, X.; Wu, N.; Zhang, S.; Gao, M.; Wang, X. Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules. Int. J. Mol. Sci. 2023, 24, 800. https://doi.org/10.3390/ijms24010800
Yao J, Li X, Wu N, Zhang S, Gao M, Wang X. Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules. International Journal of Molecular Sciences. 2023; 24(1):800. https://doi.org/10.3390/ijms24010800
Chicago/Turabian StyleYao, Jin, Xingmei Li, Na Wu, Songlin Zhang, Min Gao, and Xiping Wang. 2023. "Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules" International Journal of Molecular Sciences 24, no. 1: 800. https://doi.org/10.3390/ijms24010800