Sep 02, 2021
- Jonathan Houseley,
- Cristina Cruz
- Springer Nature Books
External link: https://doi.org/10.1007/978-1-4939-9822-7_5
Protocol Citation: Jonathan Houseley, Cristina Cruz 2021. Hybridization of DNA Oligonucleotide Probes. protocols.io https://dx.doi.org/10.17504/protocols.io.bns6mehe
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 22, 2020
Last Modified: September 02, 2021
Protocol Integer ID: 43582
Keywords: lncRNA, ncRNA, northern blot, hybridization, probes,
Abstract
Over the past decade a plethora of noncoding RNAs (ncRNAs) have been identified, initiating an explosion in RNA research. Although RNA sequencing methods provide unsurpassed insights into ncRNA distribution and expression, detailed information on structure and processing are harder to extract from sequence data. In contrast, northern blotting methods provide uniquely detailed insights into complex RNA populations but are rarely employed outside specialist RNA research groups. Such techniques are generally considered difficult for nonspecialists, which is unfortunate as substantial technical advances in the past few decades have solved the major challenges. Here we present simple, reproducible and highly robust protocols for separating glyoxylated RNA on agarose gels and heat denatured RNA on polyacrylamide–urea gels using standard laboratory electrophoresis equipment. We also provide reliable transfer and hybridization protocols that do not require optimization for most applications. Together, these should allow any molecular biology lab to elucidate the structure and processing of ncRNAs of interest.
Introduction
Northern blotting methods allow for simultaneous quantification and molecular weight determination of RNA. Although superseded by qPCR and sequencing methods for routine mRNA quantification, northern blotting is the method of choice when complex mixtures of overlapping species are under investigation. This is particularly true when studying RNA processing by complexes such as the exosome, and generally aids in resolving the behavior of differentially expressed RNA isoforms. Historically, northern analysis has been something of a black art; running a high-quality formaldehyde gel required substantial skill and a little luck, while radioactive probing of RNA membranes often resulted in terrible cross-hybridization and invisible signals. Fortunately, technology has moved on such that modern northern analysis methods are simple and robust.
Electrophoretic separation of single-stranded RNA is more complex than double-stranded DNA as RNA forms strong secondary structures that impede separation by molecular weight in a gel matrix. For analysis of high molecular weight RNA in agarose gels, chemical modification of guanine is the preferred method to melt secondary structure, which disrupts C:G base pairing and allows single stranded RNA to migrate according to size. Although formaldehyde has been widely used for this purpose [1], it is not ideal due to issues with sample migration and batch-to-batch variation. Furthermore formaldehyde gels release toxic formaldehyde gas; this did not overly concern early investigators eager to replace the hideously toxic denaturant methyl mercury used in the first northern blotting protocols [2,3], but is clearly a problem in modern labs. To circumvent these issues glyoxal was long ago suggested as an effective RNA denaturant [4], but originally required technically awkward buffer recirculation. This problem was solved by the introduction of BPTE running buffer, which allows agarose gels of glyoxylated RNA to be run with no more difficulty than a normal DNA agarose gel [5].
High-resolution separation of small RNA fragments (~20–250 bp) is best performed on denaturing acrylamide gels, which rely on heat and urea rather than chemical modification to prevent secondary structure formation. The technique is identical to traditional sequencing gel electrophoresis [6], however, the apparatus used for sequencing is not practical for northern blotting and standard protein gel electrophoresis systems are well-suited for this purpose. Helpfully, many of the complications inherent to running a high quality sequencing gel can be safely ignored unless base-pair resolution is required.
Separated RNA is transferred to a membrane by capillary transfer for agarose gels or using an electroblotting system for acrylamide gels [7,8,9]. Various different membranes and transfer conditions have been described but we find charged nylon membrane best for all standard applications [10], and observe little difference between transfer methods. Similarly, many combinations of probes and hybridization buffers can be used to detect RNA species, each having their own strengths and weaknesses. Here we provide a protocol for using RNA probes transcribed from PCR products; in our hands these are both the most reliable and the most sensitive, and as such are the probe of choice for new users [11,12,13,14]. We also provide probing conditions for use of synthetic oligonucleotides, which are widely used in RNA processing analysis since they provide unparalleled resolution of intermediates, and for random-primed DNA probes.
The source of RNA used for northern blotting is rarely critical as long as it is of high quality (see Note 1), and therefore in this chapter we focus purely on the gel systems and hybridization methods.
Guidelines
RNA Handling and RNase-Free Technique
The difficulties in handling RNA are often overemphasized. In our hands, most of the cumbersome procedures recommended for avoiding RNase contamination seem to be dispensable. We use tubes and water specifically designated for RNA work, but take few other precautions. Solutions for gels and blotting are made with milliQ water in normal lab bottles with standard chemicals weighed in disposable weighing boats. We do not DEPC treat water or solutions, or use RNase decontaminating sprays or wipes routinely. It is good to have RNase ZAP in the laboratory to clean occasional contaminations or to wipe for example tissue grinding tools that will be in direct contact with the sample. For the final resuspension of RNA samples, we use commercially available nuclease free water. On first use, electrophoresis tanks can be rendered RNase-free by treating with 3% H2O2for 10 min, then rinsed with milliQ water, then set aside for RNA work if required. Blotting and hybridization can be carried out in normal laboratory trays and glassware. We use certified RNase-free filter tips and set aside a specific set of pipettes for handling stocks of RNase and RNase-containing solutions like plasmid miniprep resuspension buffer. Our bodies are a good source of RNase contamination, so care should be taken to avoid touching the inside of the lids when handling tubes. After assembling reagents, solutions and equipment for northern blotting, it is advisable to run a test gel using RNA of known quality before handling precious samples; ribosomal RNA bands should be clearly resolved and the higher molecular weight band (28S in mammals, 25S in yeast) should be brighter than the lower band (18S). This will confirm that the reagents are sufficiently RNase free.
Conversely, nonenzymatic mechanisms of RNA degradation need to be considered: RNA hydrolysis is catalyzed by alkaline pH and/or divalent cations, particularly with increasing temperature. Therefore, store RNA in water as opposed to TE (pH 8) or similar and beware of reaction conditions involving divalent cations and heat. DNase treatment can be a problem as all DNase I buffers contain magnesium, and DNase treatment is rarely necessary for northern blots as high molecular weight genomic DNA usually resolves far from the bands of interest.
Materials
RNA Handling and RNase-Free Technique
1. A source of RNase-free milliQ water.
2. Commercially available nuclease free water.
3. Certified RNase-free filter tips.
4. 1.5 mL microfuge tubes specifically designated for RNA work.
5. A set of pipettes that are RNase-free.
6. RNaseZAP (Sigma) or similar.
7. 3% H2O2.
Hybridization of DNA Oligonucleotide Probes
1. Desalted oligonucleotide probe (synthesized by Sigma-Genosys or equivalent).
Note
We have occasionally observed that desalted oligonucleotides do not label well due to unknown contaminants from the manufacturing process. This seems occur at random. Because of this, we routinely purify new oligonucleotide batches using a Nucleotide Removal Kit (Qiagen). Other oligonucleotide manufacturers may not have similar problems, and ordering HPLC-purified oligonucleotides may also solve this problem.
2. QIAquick Nucleotide Removal Kit (QIAGEN) or similar.
3. 10 U/μL T4 polynucleotide kinase and buffer (NEB).
4. 100 mM DTT.
5. 3000 Ci/mMol [γ-32P] ATP, 185 MBq/mL.
Note
32P sources do not need to be particularly fresh. We use sources up to 6 weeks past the activity date with no problem, or even more for high signals. The exposure time required obviously increases, but the output is similar.
6. Radioactive workroom and protective equipment.
7. mini Quick Spin Oligo Columns by Roche or similar.
8. Hybridization bottles and oven.
9. ULTRAHyb Oligo ultrasensitive hybridization buffer by Ambion (Thermo Fisher Scientific).
10. 6× SSC.
11. 2× SSC 0.1% SDS.
12. Typhoon Storage Phosphorimager FLA7000 (GE) or equivalent.
13. Storage phosphor screen and exposure cassette.
14. FLA image eraser (GE) or equivalent.
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
IMPORTANT: Make sure you have appropriate training to work with radioactivity under the local rules and legislation for your institution, and perform all radioactive work in the designated area.
Before start
It is recommended to read through the guidelines before starting any work with RNA.
Hybridization of DNA Oligonucleotide Probes
Hybridization of DNA Oligonucleotide Probes
2h 50m
2h 50m
Note
Oligonucleotide probes are normally 20–45 nt, 40% GC, this protocol is designed for 30–45 nt DNA probes. They are very good for detecting abundant targets by northern blot and allow precise dissection of processing intermediates, but not all probes label or hybridize well for reasons that remain unclear. Oligonucleotide probes can be stripped off the membrane very easily which is useful. Probes should not be designed with a C at the 5′ end as this inhibits labeling. Clean the oligonucleotide probe using a QIAquick Nucleotide Removal Kit before labeling.
Note
RNA or LNA oligos can also be employed to improve specificity but the hybridization conditions will need to be optimised.
Ensure that the hybridization bottles and internal seals are in good condition to avoid radioactive buffer leaking during hybridization.
Prewarm a bottle of ULTRAHyb Oligo to 68 °C until the SDS fully dissolves (00:15:00 –00:20:00 ).
35m
Wet the membrane with 0.1× SSC 0.1% SDS. Roll it up with the RNA facing inward, drop it into the hybridization bottle and slowly turn the bottle to unroll the membrane against the side of the bottle.
Note
A Stripette can be used to help with this. Push out any obvious air bubbles. Put bottles in the oven so that the visible edge of the membrane is moving with the direction of rotation; if the membrane rolls up into a thin cigar during hybridization or washing then it was the wrong way round. When using a hybridization oven with rotisserie, always be sure to balance the bottles, including the volume of liquid inside. Two membranes can be hybridized in the same bottle, one facing the glass and the other facing the inside of the bottle.
Prehybridize for 01:00:00 at 42 °C with 7.5 mL UltraHyb Oligo .
Note
This is an important step so do not feel tempted to skip it or shorten it. It allows blocking agents in the hybridization buffer to occupy nonspecific binding sites.
1h
Mix in this order:
5 picomolar (pM) oligonucleotide
1.5 µL 10× T4 polynucleotide kinase buffer
1.5 µL 100 mM DTT
Water to 9 μL
1 µL T4 polynucleotide kinase
5 µL [γ-32P] ATP 3000 Ci/mmol 185 MBq/mL
Note
The amount of oligonucleotide is important. The molarity of most radionucleotides is very low (this can be calculated for any source as the activity/mmol and the activity/unit volume are provided—for the recommended [γ-32P] ATP, the molarity is 1.67 pM/μL on the activity date). Aim to have less oligonucleotide than [γ-32P] ATP—increasing the amount of oligo will seem to improve the incorporation, but will increase the amount of unlabeled oligonucleotide that competes for hybridization much more. In other words, this protocol produces a small amount of very highly labelled probe which is best for hybridization.
Incubate 01:00:00 at 37 °C .
Note
In our hands 2 h or more of labeling is fine.
1h
Clean the probe through a column as for an RNA probe (as detailed in the following substeps), but use a mini Quick Spin Oligo column as oligonucleotides are retained in normal mini Quick spin columns. Incorporation can be variable.
Flick a mini Quick Spin column inverted and then correct way up to get the sepharose to the bottom, remove top then bottom cap (see product instructions).
Place the column in a 2 mL microcentrifuge tube, spin at 1000 x g, 00:00:30 , discard tube and place the column in a new 1.5 mL screw cap tube.
Dilute the probe with water to 50 µL and then pipette carefully on to the centre of the sepharose matrix in the column.
Centrifuge at 1000 x g, 00:04:00 .
Test incorporation by briefly holding the probe and column at equal distances from the Geiger counter. Use tweezers for this. At least 50% of the label should be incorporated, which means the Geiger counter will read the same signal from both column and probe. Discard the column to the radioactive waste.
Add the probe to the hybridization bottle while vertical so that it falls directly into the hybridization buffer and incubate Overnight at 42 °C .
Pour the probe into a 50 mL tube (Falcon/Corning type) and store at -20 °C .
Note
RNA probes and oligonucleotide probes can be reused up to 2 times (a total use of 3 times) but only over a week or two taking into account the half-life of the isotope. To reuse a probe, prehybridize the membrane with half the normal quantity of hybridization buffer, meanwhile defrost the probe by letting the tube stand in a beaker of hot water for 20 min then pour the probe into the hybridization bottle.
See Section "Hybridization of RNA Probes" for washing and exposing directions:
Perform a single low stringency wash with 6× SSC for 00:10:00 at 42 °C .
10m
Perform a single high stringency wash with preheated 2× SSC 0.1% SDS at 42 °C for 00:10:00 .
10m
Perform one last wash for 00:10:00 with 50 mL 6× SSC at Room temperature .
Note
Washing twice for 30 min each with 2× SSC 0.5% SDS at 42 °C can improve signal-to-noise but can also reduce the signal for some probes. The easiest approach when using a new probe is to follow the low stringency washing protocol, expose the membrane and if background is unacceptable rewash using these more stringent conditions.
10m