TERRA RNA-FISH

RNA FISH protocol for PNA-Tel probe

• Wash cells with PBS (2 times)
• 30'' Cytobuffer+RNasin (add 10 ul RNasin to 10 ml solution)
• 30'' Cytobuffer plus Triton+RNasin
• 30'' Cytobuffer+RNasin
• 10' 4% PFA+10% Acetic Acid
• 2xrinse PBS
• 3xrinse with 70% EtOH ice cold (samples can be kept in this buffer until 3-months)
• 3' 80% EtOH ice cold
• 3' 95% EtOH ice cold
• 3' 100% EtOH ice cold
• Let slide air dry (very briefly, aprox. 10 seconds)
• Add 8ul PNA-Tel probe (8 ul PNA probe* from stock at 0,5 ng/ul in hybridization buffer), seal with cover slip and incubate in wet chamber overnight at 37℃. 
• 2x15' [2xSSC, 50% Formamide], 39℃ (prewarm buffer prior to washes)
• 10' 2xSSC, 39℃ (prewarm buffer prior to washes)
• 10' 1xSSC, 39℃ (prewarm buffer prior to washes)
• 5' 4xSSC, room temperature
• 5 4xSSC, 0.1% Tween 20
• 5' 4xSSC
• Mount samples with Prolong (contains DAPI)

Cytobuffer (500ml)

100 mM NaCl (10ml of 5M NaCl)

300 mM Sucrose (51.3 gr of powder)

3 mM MgCl₂ (1.5ml of 1M MgCl₂)

10 mM Pipes pH 6.8* (5ml of 1M Pipes)

Cytobuffer+Triton X100 (500 ml)

100 mM NaCl

300 mM Sucrose

3 mM MgCl₂

10 mM Pipes pH 6.8

0.5% Triton X100 (2.5ml of Triton X-100)

Hybridization buffer

50% Formamide

10% Dextrane sulfate

2xSSC