Northern blot analysis

Northern blot is one of a series of blotting techniques used to transfer RNA onto a carrier to quantify RNA expression. The Northern blot takes its name from its predecessor, the Southern blot, which was named after the biologist Edwin Southern. In Northern blot, RNA is separated in a denaturing agarose gel, transferred by capillary transfer to a nitrocellulose membrane (vacuum blot) and fixed by UV crosslinking. The RNA of interest is identified by hybridization with a specific RNA probe. In order to prevent the contamination with RNases all the solutions used in Northern blot were autoclaved and the apparatus used for electrophoresis were kept in 3% H2O2

for 1 h before use.

3.2.14.1 Denaturation of RNA

The RNA samples to be analyzed on a blot having 30 µg and 16.5 µl of loading buffer were denatured at 68 ˚C for 20 min and subsequently cooled on ice for 2 min. The denatured RNA samples were loaded on a denaturing formaldehyde agarose gel along with 5 µl of sample buffer.

Loading buffer Final concentration

Formamide 15 ml 66.6%

14.3 X MOPS 2.1 ml 26.7 mM

37% formaldehyde 5.4 ml 8.9%

The buffer was aliquoted and stored at -20 ˚C.

14.3 X MOPS Final concentration

MOPS 5.93 g 286 mM

NaAc 0.58 g 7.1 mM

EDTA 0.5 g 1.3 mM

DEPC-H2O to 100 ml

The pH was adjusted with NaOH to 7.0 and the solution was autoclaved.

Sample buffer Final concentration

Glycerin 5 ml 50%

0.5 M EDTA 20 µl 1 mM

Bromophenol blue 10 mg 0.1%

DEPC-H2O to 10 ml The buffer was aliquoted and stored at -20 ˚C.

3.2.14.2 Denaturing agarose gel electrophoresis of RNA

Denatured RNA samples were resolved on a 1.5% formaldehyde agarose gel and electrophoresis was performed at 110 volts for 2 h. For preparation of a 1.5% gel 1.2 g agarose were dissolved in 65 ml DEPC-H2O. Then 8 ml of 10 X MOPS and 6.7 ml of formaldehyde were added. After mixing, the gel was poured into the prepared gel plate (10 X 14 cm) and allowed to solidify at room temperature. Then the gel was transferred into an electrophoresis chamber filled with 1 X MOPS buffer.

After gel electrophoresis the formaldehyde was washed out by shaking the gel in 150 ml of a 1%

glycine solution for 20 min. To visualize the RNA under UV light (254 nm) 5 µl of ethidium bromide was added and the gel was further incubated for 5 more min. Two main bands corresponding to 28S and 18S ribosomal RNA were visible under UV light, then the gal was photographed. Finally, the gel was washed in 20XSSC for 20 min before blotting onto a nylon membrane.

10 X MOPS Final concentration

MOPS 41.9 g 200 mM

NaAc 4.1 g 50 mM

EDTA 3.7 g 10 mM

DEPC-H2O to 1 l

The pH was adjusted to 7.0 with NaOH. The solution was autoclaved.

3.2.14.3 RNA blotting onto nylon membrane

The resolved RNA in the gel was transferred onto a nylon membrane (Hybond N⁺, Amersham) by capillary blotting. The nylon membrane was presoaked in 2 X SSC for 10 min. The blotting equipment was prepared in the following way; A plastic tray filled with 700 ml of 10 X SSC buffer, a flat surfaced glass stand was fixed above the buffer level and covered with three layers of 3 mm thick filter paper (presaturated in 20 X SSC) with their ends dipped in the buffer underneath; RNA gel with the upper side downwards and surrounded by foil stripes to prevent drying out of papers; the nylon membrane was put on top of the gel; 3 layers of 3 mm filter papers with the size of the gel; approx. 10 cm of paper towels; an equally distributed weight of approx. 1 kg. The capillary blotting was carried out for at least 16-18 h, the arrangement was dismantled and the RNA was irreversibly cross-linked by UV radiation in an UV cross-linker (Stratagene) for 2 min at 1200 µJ.

3.2.14.4 Hybridization of RNA with digoxigenin-labeled RNA probes

The RNA blot was placed in a hybridization glass tube and pre-hybridized with 10 ml of hybridization solution at 65-68˚C in a hybridization oven. After 1 h of incubation, the pre-hybridization solution was replaced with the 10 ml of pre-hybridization solution containing 150 ng of digoxigenin-labeled antisense RNA probe. The hybridization was performed at 65˚C overnight.

The unbound probe was removed by washing with 2 X SSC / 0.1% SDS for 2 X 5 min and 0.1 X SSC / 0.1% SDS for 2 X 15 min at 65˚C.

Pre- and hybridization solutions Final Concentration

Deionized formamide 12.5 ml 50% (v/v)

10 % blocking reagent 6 ml 2.5% (w/v)

20 % SDS 25 µl 0.02% (w/v)

10 % N-lauroylsarcosine 250 µl 0.1% (w/v)

20 X SSC 6.25 ml 5 X

The solution was stored at 4˚C.

3.2.14.5 Detection and quantification

The detection of hybrids between the RNA of interest and the digoxigenin-labeled asRNA was performed using anti-digoxigenin antibodies conjugated to alkaline phosphotase (enzyme immunoassay). During the following dephosphorylation of dinatrium 3-(4-methoxyspiro[1,2-dioxetane-3,2-(5’-chloro)tricycle{3.3.1.1^3.7}decan}-4-yl)-phenylphosphat (CSPD) by alkaline phosphatase a chemiluminescent unstable product was formed which produced light of 477 nm.

This light signal can be recorded on X-ray films. The quantification was made densitometrically.

The detection was performed with the DIG nucleic acid detection kit (Roche) according to the manufacturer instructions. After hybridization and posthybridization washes of the nylon membrane it was equilibrated in 1 X maleic acid buffer for 1 min. To prevent nonspecific binding of the DIG antibody to the membrane it was washed for 30 min in 100 ml 1% blocking reagent (10% blocking reagent diluted to 1% with 1 X maleic acid buffer). Then, the nylon membrane was incubated for 30 min at room temperature in 20-30 ml diluted antibody solution (anti-digoxigenin Fab fragments, conjugated to alkaline phosphatase, in 1% blocking reagent at a concentration of 75 mU/ml = 1:10000 dilution which is equal to 5 µl antibody in 50 ml 1%

blocking reagent). The unbound antibody was removed by washing in maleic acid buffer, containing 0.3% Tween – 2 X 15 min. The washed membrane was equilibrated in buffer 3 for 1-2 min and wrapped in transparent foil along with the diluted CSPD solution for 5 min. (CSPD in buffer 3 at a concentration of 2.5 mM =1:1000 = 1 µl CSPD in 1 ml buffer 3). The liquid was completely removed and the membrane was incubated for 10 min at 37˚C. Then the membrane was exposed to a X-ray film for 30-90 min.

For quantification of the RNA bands, IMAGE J software was used.

Buffer 1 (Maleic acid buffer) Final concentration

Maleic acid 11.61 g 0.1 M

NaCl 8.78 g 0.15 M

H2O to 1 l

The pH was adjusted to 7.5 with solid NaOH. The solution was autoclaved.

Buffer 2 450 ml of buffer 1

50 ml 10 X blocking reagent

Buffer 3 Final concentration

Tris 12.11 g 0.1 M

NaCl 5.84 g 0.1 M

MgCl2 10.17 g 50 mM

H2O to 1 l

The pH was adjusted with HCl to 9.5. Then the buffer was autoclaved and sterile filtered, MgCl2

was added at the end.