Poly(A) RNA in situ localization

Protocol from:(Zhizhong Gong et al. 2005)(Parry et al. 2006)

 

Oligo d(T) should be 45-50nt long and labeled in 5’. Do not use fluorophore in the red range has they would be mask by chlorophyll. Alexa-488 is a good choice (Invitrogen).

 

Fixation buffer:

120 mM NaCl

7 mM Na2HPO4

3 mM NaH2PO4

2.7 mM KCl

0.1% Tween 20

80 mM EGTA

5% formaldehyde

10% DMSO

 


1.      2-7 days plates grown seedlings are immersed in 10 ml Fixation cocktail (50% fixation buffer + 50% heptane), glass container (scintillation vial) and gently agitate for 30 min at RT.

2.      Dehydrate twice 5 minutes in methanol 100%

3.      Dehydrate three times 5 minutes in ethanol 100%

4.      Incubate 30 min in ethanol:xylene (50:50)

5.     

DAY1

Approx 4h

Wash twice times 5 minutes in ethanol

6.      Wash twice 5 min with methanol

7.      Incubate 5 min in methanol: fixation buffer without formaldehyde (50:50)

8.      Incubate 30 min in fixation buffer with formaldehyde

9.      Rinse 5 min twice with fixation buffer without formaldehyde

10.  Rinse 5 min once with 1 ml Hybridization buffer (Sigma).

11.  Change for new (1 ml) hyb buffer and pre-hybridized at 50°C/1h.

12.  Add 5 pmol of 5’-labeled oligo d(T).

13.  Hybridize at 50°C overnight in the dark.

14.  Mount slides in water, and observe using the confocal.

 

 

 

 

Gong, Zhizhong, Chun-Hai Dong, Hojoung Lee, Jianhua Zhu, Liming Xiong, Deming Gong, Becky Stevenson, and Jian-Kang Zhu. 2005. A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. The Plant Cell 17, no. 1 (January): 256-67. doi:10.1105/tpc.104.027557.

Parry, Geraint, Sally Ward, Alex Cernac, Sunethra Dharmasiri, and Mark Estelle. 2006. The Arabidopsis SUPPRESSOR OF AUXIN RESISTANCE proteins are nucleoporins with an important role in hormone signaling and development. The Plant Cell 18, no. 7 (July): 1590-603. doi:10.1105/tpc.106.041566.