Abstract
The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.
Highlights
To cite this version: Maëlle Jaouannet, Chinh Nghia Nguyen, Michaël Quentin, Stéphanie Jaubert-Possamai, Marie-Noëlle Rosso, et al
We adapted the In situ hybridization (ISH) technique to the plant parasite Meloidogyne spp. to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues
We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure
Summary
PCR-grade dNTPs (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297117) 13. TE buffer pH 8 (10 mM Tris-HCl, 1 mM EDTA) (e.g., Sigma-Aldrich, catalog number: 93283) 18. RNaseZap® RNase Decontamination Solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780) 19. 10x PBS (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9624). Proteinase K 20 mg/ml (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2546) 36. SSC buffer 20x (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9770) 38. SDS (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15553027) 40. EDTA pH 8 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9261) 42. Salmon sperm DNA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15632011) 43. TRNA from baker’s yeast (Sigma-Aldrich, catalog number: R8759, type X-SA) 44. Nuclease-free water (not DEPC-Treated; Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9938) 51.
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