Mar 25, 2020
Hybridization and detection of small RNA samples with DIG-labeled probes
- Alice Pawlowski1
- 1Institute of Synthetic Microbiology, CEPLAS, Heinrich Heine University Duesseldorf
- Axmann Lab
Protocol Citation: Alice Pawlowski 2020. Hybridization and detection of small RNA samples with DIG-labeled probes. protocols.io https://dx.doi.org/10.17504/protocols.io.us6ewhe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2018
Last Modified: March 25, 2020
Protocol Integer ID: 16958
Abstract
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).
It is adjusted to Northern Blots with membrane size of around 9 x 10 cm2 .
Attachments
Guidelines
Pay attention to the standard precautions needed for working with RNA (e.g. always ware cloves; clean pipettes, surfaces, trays with decontaminant (e.g. RNase away), use RNase free solutions)
Materials
MATERIALS
DIG Easy Hyb™ GranulesRocheCatalog #11796895001
Blocking ReagentRocheCatalog #11096176001
Anti-Digoxigenin-AP Fab fragmentsRocheCatalog #11093274910
ROTI®Stock 20x SSCCarl RothCatalog #1054.1
ROTI®Stock 20 % SDSCarl RothCatalog #1057.1
Nuclease free water DEPC treatedCarl RothCatalog #T143.3
CDP-Star® ready-to-useRocheCatalog #12041677001
square petri dishes 120 x 120 x 17 mm PSgreiner bio-oneCatalog #688161
Hybridization:
DIG Easy Hyb
- reconstitute DIG Easy Hyb Granules with 64 ml sterile, nuclease free water (add 32 ml of water to the bottle and incubate at 37°C with shaking until powder is solved, then add another 32 ml of water), takes time!
10 x TBE buffer
- 108 g/l Tris
- 55 g/l Boric acid
- 100 ml/l 0.5 M EDTA, pH 8.0
- Hybridization oven
- Hybridization tubes 150 mm length
- Thermoblock
Stringency washes:
2 x SSC + 0.1 % SDS
prepare freshly from 20xSSC and 20% SDS stocksolution
(5 ml 20xSSC and 250 µl 20% SDS in 50 ml Water)
0.1 x SSC, 0.1 % SDS:
5 ml 20 x SSC and 5 ml 20 % SDS in 1 L water
(stored at RT; solution might get slightly turbid due to SDS precipitation, but gets clear when heated)
- hybridization oven
- table shaker
- square petridishes (120 x120 x 17 mm)
Immunological detection
Maleic acid buffer (Dilution of blocking solution)
- 0.1 M maleic acid
- 0.15 M NaCl
adjust with solid NaOH to pH 7.5 (20°C)
prepare 1 L of 2 x maleic acid buffer (23.2 g maleic acid, 17.54 g NaCl), add NaOH plates to pH6.0 - 6.5 , afterwards proceed carefully with 5 M and 1 M NaOH, because pH starts to rise rapidly when getting near 7.5!
Solid NaOH takes a while to get into solution, thus buffer preparation takes time!
Autoclave
Washing buffer (removal of unspecific bound atibody)
Maleic acid buffer + 0.3 % Tween 20
prepare freshly, e.g. 100 ml 2 x maleic acid buffer + 100 ml H2O + 0.6 ml 20% Tween
Detection buffer (pH adjustment )
- 0.1 M Tris-HCl
- 0.1 M NaCl
adjust to pH 9.5 (20 °C)
prepare a 10 x stock solution (58.44g NaCl, 121.14 g Trizma-base, pH9.5) and autoclave
10 x Blocking solution (blocking of unspecific binding sites on the membrane)
- prepare a 10% (w/v) solution of blocking reagent powder in maleic acid buffer, e.g add 10 g to 100 ml maleic acid buffer in a 250 ml Schott flask
- dissolve the powder by succesive cycles of heating in a microwave
- 10 x blocking solution is stored at 4 - 8°C
CDP-Star, ready-to use solution
- store bottles at 4-8 °C
Before start
- make sure that you have all buffers and solutions prepared; reconstitution of DIG Easy Hyb granules and preparation of maleic acid buffer and 10 x blocking solution is time consuming!
- make sure that you have your DIG-labled probes ready: to receive sufficient amount of probe (~ 1 µg), in vitro transcription of small RNA fragments takes 4-5 h!
Hybridization
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 buffer
- pre-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 tube
- membrane 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
Overnight
stringency washes
stringency washes
All washing steps are done with 25 ml volume
- place 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 oven
- clean 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
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 buffer
- prepare 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 use
- pipet 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 probe
- wash 2 x 15 min in Washing buffer in a fresh petridish
- eqilibrate the menbrane 2 - 5 min in Detection buffer
- place 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 soaked
- Immediately cover the membrane with the second sheet to spread the substrate evenly and without air bubbles over the membrane
- incubate for 5 min at RT; for trRNA detection, a prolonged (1 h) incubation time led to stronger signals
- squeeze 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
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 signal
- take 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
Overnight
- proceed with the protocol steps 2-3
- no 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 cm
- signal is detected after 10 - 20 sec exposure time