TITLE: Hybridization and detection of small RNA samples with DIG-labeled probes
AUTHORS: Alice Pawlowski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to hybridize small RNAs (50 - 200 nt) - either from total RNA isolates or from in vitro transcripts - with digoxigenein-labeled probes, followed by detection with chemiluminescence using CDP-star.  The protocol follows mainly the instructions given in the DIG Northern Starter Kit, Version 8.0 (Roche). </div><div class = "text-block"><span>It is adjusted to  Northern Blots with membrane size of around 9 x 10 cm</span><span style = "vertical-align:super;">2 </span><span style = "vertical-align:super;">.</span></div></div>

[STEPS]
?. [Hybridization]
prewarm an appropriate volume of DIG Easy Hyb to 62 °C (5 ml per hybridization tube) wet the Northern Blot membrane (9 x 10 cm2 size) in 0.5 x TBE bufferpre-hybridize the membrane with prewarmed DIG Easy Hyb solution in a hybridization tube ( role the membrane with the RNA facing inside) for 30 min at 62 °C  in an hybridization oven with constant rotation; don't forget to use a balance tube when you have just one tubemembrane should move freely ( use just 1 membrane per tube to avoid stacking of membranes) denature the DIG-labeled probe (in vitro transcript of trRNA sequence) for 5 min at 95°C in a thermoblock, cool down on ice and mix with 5 ml prewarmed DIG-Easy Hyb remove pre-hybridization solution and add probe/hybridization mixture to the membrane hybridize o/n at 62 °C
?. [stringency washes]
All washing steps are done with 25 ml volumeplace membrane (RNA facing upwards) in a cleaned square petridish (120 x 120 x 17 mm)wash 2 x 5 min in 2 x SSC + 0.1% SDS at RT under constant agitation on a table shaker (90 rpm)meanwhile prewarm an appropriate volume of 0.1 x SSC + 0.1 SDS at 68 °C in the hybridization ovenclean the hybridization tube and re-use it to wash the membrane for 2 x 15 min at 68 °C with constant rotation
?. [Immunological detection of trRNA]
All incubations are performed at RT in a square petridish (120 x 120 x 17 ml), with 25 ml solution each, on a table shaker (90 rpm)after stringency washes, rinse the membrane shortly (1-5 min) in Washing bufferprepare a 5 x blocking solution by diluting 10 x Blocking solution 1: 2 in maleic acid buffer (50 ml falcon tube, always prepare fresh)incubate for at least 30 min in 5 x blocking solution in a fresh, cleaned petridish centrifuge the anti-digoxigenin-AP antibody for 5 min at 10 000 rpm and 4 °C prior to usepipet the necessary amount carefully from the surface dilute antibody 1 : 10000 (75 mU/ml) in fresh 5 x blocking solution (5 µl in 50 ml 5 x blocking solution)incubate the membrane for 30 min in Antibody solution to bind the antibody to the DIG-labeled probewash 2 x 15 min in Washing buffer in a fresh petridisheqilibrate the menbrane 2 - 5 min in Detection bufferplace the membrane (with RNA side facing up) on a development folder or hybridization bag; we put the membrane between the sheets of a transparent, clear disposable waste bag (Sekuroka, 300 x 200 mm, 40 µm, PP)lift one sheet up and quickly apply approx. 1 ml CDP-Star, ready-to-use solution out of the dropper bottle until the membrane is evenly soakedImmediately cover the membrane with the second sheet to spread the substrate evenly and without air bubbles over the membraneincubate for 5 min at RT; for trRNA detection, a prolonged (1 h) incubation time led to stronger signalssqueeze out excess liquid, but keep the membrane damp; drying of the membrane will result in dark background!signals were detected by using the Bio-Rad Gel Doc imaging system, program chemiluminescence- high sensitivity, image area same as for the corresponding PAA-gel (13.4 x 10.0 cm) and exposure times 20 - 200 sec (trRNA)rinse the membrane briefly in 0.5 x TBE, dry completely and store between the sheets of Whatman paper for hybridization with 5S-RNA probe (no stripping of membrane required)
?. [Immunological detection of 5S-RNA]
membranes hybridized with trRNA probes could be reprobed with the 5S-RNA specific probe without stripping of the membrane; the 5S-RNA signal is extremely strong and therefore detectable at exposure times too short for the detection of the trRNA signaltake the dried membrane and start with the hybridization protocol (Step 1)hybridize the membrane with the in vitro transcript of the 5S-RNA sequence at 62 °C  proceed with the protocol steps 2-3no incubation necessary after application of CDP-Star ready-to-use solution, immediately expose the membrane to the Gel Doc imager, program chemiluminescence- high sensitivity, image area 13.4 x 10.0 cmsignal is detected after 10 - 20 sec exposure timeproceed with the protocol steps 2-3no incubation necessary after application of CDP-Star ready-to-use solution, immediately expose the membrane to the Gel Doc imager, program chemiluminescence- high sensitivity, image area 13.4 x 10.0 cmsignal is detected after 10 - 20 sec exposure time