TITLE: Hybridization of DNA Oligonucleotide Probes
AUTHORS: Jonathan Houseley, Cristina Cruz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Introduction</span></div><br/><div class = "text-block">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 Although superseded by qPCR and sequencing methods for routine mRNA quantification, northern blotting is the method of choice when complex mixtures of overlapping species are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.</div><div class = "text-block"><span>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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">1</span></a><span>], 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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR2" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">2</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR3" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">3</span></a><span>], but is clearly a problem in modern labs. To circumvent these issues glyoxal was long ago suggested as an effective RNA denaturant [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR4" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">4</span></a><span>], 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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR5" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">5</span></a><span>].</span></div><div class = "text-block"><span>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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">6</span></a><span>], 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.</span></div><div class = "text-block"><span>Separated RNA is transferred to a membrane by capillary transfer for agarose gels or using an electroblotting system for acrylamide gels [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR7" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">7</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR8" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">8</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">9</span></a><span>]. Various different membranes and transfer conditions have been described but we find charged nylon membrane best for all standard applications [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR10" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">10</span></a><span>], 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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR11" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">11</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR12" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">12</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR13" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">13</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9822-7_5#CR14" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">14</span></a><span>]. 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.</span></div><div class = "text-block"><span>The source of RNA used for northern blotting is rarely critical as long as it is of high quality (</span><span style = "font-style:italic;">see </span><span style = "font-weight:bold;">Note 1</span><span>), and therefore in this chapter we focus purely on the gel systems and hybridization methods.</span></div></div>

[STEPS]
?. [Hybridization of DNA Oligonucleotide Probes]
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.
RNA or LNA oligos can also be employed to improve specificity but the hybridization conditions will need to be optimised.
?. [Hybridization of DNA Oligonucleotide Probes]
Ensure that the hybridization bottles and internal seals are in good condition to avoid radioactive buffer leaking during hybridization.
?. [Hybridization of DNA Oligonucleotide Probes]
Prewarm a bottle of ULTRAHyb Oligo to  until the SDS fully dissolves (–).
68 °C
?. [Hybridization of DNA Oligonucleotide Probes]
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.
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.
?. [Hybridization of DNA Oligonucleotide Probes]
Prehybridize for  at  with .
42 °C
[UltraHyb Oligo]
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.
?. [Hybridization of DNA Oligonucleotide Probes]
Mix in this order:Water to 9 μL
[oligonucleotide]
[10× T4 polynucleotide kinase buffer]
[100 mM DTT]
[T4 polynucleotide kinase]
[[γ-32P] ATP 3000 Ci/mmol 185 MBq/mL]
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.
?. [Hybridization of DNA Oligonucleotide Probes]
Incubate  at .
37 °C
In our hands 2 h or more of labeling is fine.
?. [Hybridization of DNA Oligonucleotide Probes]
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.
?. [Hybridization of DNA Oligonucleotide Probes]
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).
?. [Hybridization of DNA Oligonucleotide Probes]
Place the column in a 2 mL microcentrifuge tube, spin at , discard tube and place the column in a new 1.5 mL screw cap tube.
Centrifuge: 1000 34, 30 s
?. [Hybridization of DNA Oligonucleotide Probes]
Dilute the probe with water to  and then pipette carefully on to the centre of the sepharose matrix in the column.
50 µl
?. [Hybridization of DNA Oligonucleotide Probes]
Centrifuge at .
Centrifuge: 1000 34, 4 min
?. [Hybridization of DNA Oligonucleotide Probes]
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.
?. [Hybridization of DNA Oligonucleotide Probes]
Add the probe to the hybridization bottle while vertical so that it falls directly into the hybridization buffer and incubate  at .
42 °C
?. [Hybridization of DNA Oligonucleotide Probes]
Pour the probe into a 50 mL tube (Falcon/Corning type) and store at .
-20 °C
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.
?. [Hybridization of DNA Oligonucleotide Probes]
See Section "Hybridization of RNA Probes" for washing and exposing directions:
?. [Hybridization of DNA Oligonucleotide Probes]
Perform a single low stringency wash with 6× SSC for  at .
42 °C
?. [Hybridization of DNA Oligonucleotide Probes]
Perform a single high stringency wash with preheated 2× SSC 0.1% SDS at  for .
42 °C
?. [Hybridization of DNA Oligonucleotide Probes]
Perform one last wash for  with  at .
[6× SSC ]
0 Room temperature
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.