miRNA insitu detection using LNA-Oligos:
This protocol was modified from Valoczi et al., 2006, The Plant Journal 47, 140-151 by
Jacqueline Gheyselinck and Patrick Sieber.
If not indicated here, all other steps follow the standard protocol (e.g. Jeff Longs) with 4%
para-formaldehyde fixation overnight at 4C and subsequent Ethanol dehydration and
embedding in paraplast. Sectioning and mounting was also done as described and so was the
rehydration, proteinaseK digest and the acetylation step.
PROBE SYNTHESIS: labelling of LNA-Oligos:
 We use DIG Oligonucleotide 3’-End Labelling Kit, 2nd Generation from Roche (Cat.No. 03
353 575 910).
 4 µl of 25µM ath-MIR164a LNA(Exiqon), 100pmol that is.
 6µl ddH2O
 keep on ice




4 µl
4 µl
1 µl
1 µl
of
of
of
of
5xRxn buffer (1 Kit)
CoCl2 Solution (2 Kit)
DIG-ddUTP (3 Kit)
Termial Transferase (4 Kit)
 Mix and inclubate at 37C for 15min
 Stop Rxn
 1µl 0.5 µM EDTA pH 8.0
 20 µl Formamide
Use 1.5µl-4.5µl of Probe in 150µl Hyb buffer. We used 3µl for the ath-MIR164a insitus.
Use scrambled-miR from Exiqon as negative control for all miRNA insitus.
gentle shaking
(50 rpm)
CHECK PROBE
 spot on nitrocellulose filter 0,5 µl and (0.5µl dil. 1:2) of probes before adding deiformamide.
 dry on air
 UV crosslink
 wet in 1x BSA wash (Solution 2)
 blocking reagent/ 1x TBS
30 min.
 anti-DIG/ 1x TBS (1:1250; 2,5 ml) 30 min.
 wash 5 min. 2 or 3 times in 1x TBS
 3 ml 1x substrate buffer
5 min.
 add 2 ml 1x Detection solution put into dark
 stop the reaction in dw.
HYBRIDIZATION
Hybridization buffer:
 10x salts
 DEI formamide
 50% dextran sulfate
 100 mg/ml tRNA
 100x Denhardt’s
 ddH2O
125 l
500 l
250 l
12.5 l
12.5 l
100 l
1ml
Heat the probe to 80°C for 5 min (e.g. 3l Probe + 30l 50% FA), then cool on ice and spin
down before adding 120 l of the hybridization buffer. Use 150 l hybridization mix + probe
per slide and cover it with cover slip, avoid bubbles. Place the slides over a glass and this on a
support inside the box. In the bottom of the box put 50%formamide/2xSSC.
50°C o/n
Day 2: WASHING
gentle shaking
(20 rpm)
gentle shaking
(20 rpm)
50% FA/2xSSC
500ml 100% Formamide
100ml 20xSSC
400ml ddH20
 50% FA/2xSSC (200ml) 50°C 30 min.
 50% FA/2xSSC
50°C 60 min.
 50% FA/2xSSC
50°C 60 min.
 1x NTE
37°C 5 min.
 1x NTE
37°C 5 min.
 1x NTE/ RNase A (20g/ml)37°C 30 min.
 1xNTE
RT
5 min.
 50% FA/2xSSC
50°C 30 min.
 50% FA/2xSSC
50°C 30 min.
 1x SSC
RT
2 min.
 1x PBS
RT
5 min.
 1x PBS
RT
5 min.
200l (10 mg/ml)/100 ml 1x NTE
 Blocking Solution (1)
30 min.
Repeat once more
30 min
(bottom of plastic box)
 1xBSA wash solution (2)
30 min.
(bottom of plastic box)
1xBSA wash solution (2)
120 min.
+Anti-DIG 1:1250
(150 l/ slide+ cover slip) Place the slides over a glass and this on a support inside the box.
Put 1xPBS to the bottom of the box.
 1x BSA wash solution (3, no Triton!) RT 15 min.
Repeat wash step 2 more times at bottom of plastic box.
 1xDetection buffer (4)
RT
5 min (Glass box and slide holder)
 1xDetection buffer (4)
RT
5 min (Glass box and slide holder)
 Detection solution (5) (150 l/ slide + cover slip)
Place the slides over a glass and this on a
support inside the box. Put detection
buffer to the bottom of the box.
 Stop the reaction by washing slides in 1xTE
 embed in Glycerol/TE or dehydrate through the Ethanol series
as described in Jeff Longs lab protocol and embed in e.g. Cytoseal etc..
SOLUTIONS
1xTBS good for one wash with slides in tray with slide rack
25ml 1 M Tris, pH 7.5
7.5 ml 5 M NaCl
fill up to 250 ml
1xblocking solution (Solution 1)
25ml 1 M Tris, pH 7.5
7.5 ml 5 M NaCl
fill up to 250 ml
add 1.25 g blocking reagent (Roche)
mix and heat to around 60C
1xBSA wash solution with Triton (Solution 2)
10ml 1 M Tris, pH 7.5
3 ml 5 M NaCl
300 l Triton X-11 (use 5ml pipette)
fill up to 100 ml with ddH20
mix
add 1g BSA (Sigma) immediately before use
mix
1xBSA wash solution without Triton (Solution 3)
40ml 1 M Tris, pH 7.5
12 ml 5 M NaCl
fill up to 400 ml with ddH20
mix
add 4g BSA (Sigma) immediately before use
mix
1x Antibody solution for 20 slides (use 150 l per slide)
spin the antibody for 5 min at 10000 at 4C.
3750 l BSA wash solution
3-4 l anti DIG antibody (Roche) 1:1250
vortex
1x Detection buffer (4)
100 ml 1M Tris, pH9.5
50 ml 1 M MgCl2
20 ml 5 M NaCl
full up to 1 liter with ddH2O
mix
1x Detection solution (5) (use 150 l per slide)
3.5 ml Western Blue
2 l 1M Levamisole
10x PBS
Composition:
1.3 M NaCl MW = 58,44 g/mol
30 mM NaH2PO4 MW (NaH2PO4.H2O) = 137,99 g/mol
70 mM Na2HPO4 MW = 141,96 g/mol)
For 1l:
NaCl 76 g
NaH2PO4.H2O 4.14 g (or 3.6 g of NaH2PO4)
Na2HPO4 9.9 g (or 12.46 g Na2HPO4.2H2O)
Set pH to 7.0
20 x SSC
Composition:
3 M NaCl MW = 58,44 g/mol
0.3 M Na3-citrate-2 H20 MW = 294,1 g/mol
For 1 liter
NaCl 175.3 g
Na3-citrate-2 H20 88.2 g
Adjust to pH 7.0 with 1N HCL and increase vol to 1 L