Abstract
This protocol describes an effective method of in situ RT-PCR that was developed to localize specific gene expression directly in thin cross-sections of nematode feeding sites induced by the cyst nematode Heterodera schachtii (H. schachtii) or the root-knot nematode Meloidogyne incognita (M. incognita) in Arabidopsis roots using DIG (Digoxigenin-11dUTP) labeling coupled with AP (alkaline phosphatase) and nitro-blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate-based detection. This method is applicable to any other Arabidopsis root tissue.
Keywords: In situ RT-PCR, Gene expression, Syncytium, Nematode feeding site, Heterodera schachtii
Materials and Reagents
Equipment
Procedure
Precautions: RNA is very sensitive and easily degraded by RNases! Therefore always wear gloves, use RNase-free water as well as glass- and plasticware. Recommended is the use of commercially available reagents for eliminating RNase and DNA contamination from the surface of glassware or plasticware e.g. RNase away!
Notes
Recipes
Acknowledgments
This protocol is a combination of methods previously published by Koltai and Bird (2000) and Urbanczyk-Wlochniak et al. (2002) and has been additionally modified. This work was supported by grant QLK-CT-1999-01501 (‘NONEMA’) from the European Union within the 5th Framework and FWF grants P16296-B06, P16897-B06 and P21067-B12.
References
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