Detection and Visualization of Specific Gene Transcripts by in situ RT-PCR in Nematode-Infected Arabidopsis Root Tissue

Materials and Reagents

1. Arabidopsis roots
   Note: Non-infected root fragments or root fragments containing feeding sites of H. schachtii (syncytia) or M. incognita (giant-cells embedded within galls).
   
2. Ethanol
   
3. Formaldehyde
   
4. Diethylpyrocarbonate-treated water (DEPC-H₂O)
   
5. Low melting agarose
   
6. Petri dishes ( 50 mm)
   
7. 12/24-well plates
   
8. Parafilm
   
9. Superglue
   
10. RNase Away (Thermo Fisher Scientific-Molecular BioProducts, catalog number: 7005 )
    
11. DNase I, RNase-free, HC (50 U/µl) (50 U/ul, 1,000 U) ((Thermo Fisher Scientic, Fermentas, catalog number: EN0523 )
    
12. RiboLock RNase Inhibitor (40 U/µl) (RiboLock: 40 U/µl) (Thermo Fisher Scientic, Fermentas, catalog number: EO0381 )
    
13. Ethylenediaminetetraacetic acid (EDTA)
    
14. SuperScript^TM III Reverse Transcriptase Kit (Life Technologies, Invitrogen^TM, catalog number: 18080-093 )
    
15. Deoxynucleotides (dNTPs)
    
16. Bovine serum albumin (BSA)
    
17. DIG (Digoxigenin-11dUTP-alkali stable; 25 nmol/25 µl) (Roche Diagnostics, catalog number: 11093088910 )
    
18. BioTherm^TM Taq DNA Polymerase (GeneCraft, catalog number: GC-002-0100 )
    
19. Anti-Digoxigenin-AP, Fab fragments (150 U/200 µl) (Roche Diagnostics, catalog number: 11093274910 )
    
20. BCIP^®/NBT Liquid Substrate System (Sigma-Aldrich, catalog number: B1911-100 ML )
    
21. Fixation solution (see Recipes)
    
22. 10x phosphate buffered saline (PBS) (see Recipes)
    
23. 20x SSC (see Recipes)
    
24. 10x PCR buffer (see Recipes)
    
25. 10x washing buffer (see Recipes)
    

Equipment

1. Vibratome (Leica, model: VT 100 )
2. Forceps
   
3. Heating plate
   
4. Laminar flow
   
5. PCR cycler
   
6. Stereo microscope
   

Procedure