TITLE: Hybridization of RNA 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 Although superseded by qPCR and sequencing methods for routine mRNA quantification, northern blotting is the method of choice when complex mixtures of overlapping species are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 RNA Probes]
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.
?. [Hybridization of RNA Probes]
Ensure that the hybridization bottles and internal seals are in good condition to avoid radioactive buffer leaking during hybridization.
?. [Hybridization of RNA Probes]
Prewarm a bottle of ULTRAHyb to  until the SDS fully dissolves (–).
68 °C
?. [Hybridization of RNA 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 RNA Probes]
Prehybridize for  at  with .
65 °C
[ULTRAHyb]
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.
In our hands, 65 °C is the best temperature for almost all RNA probes. However, if cross-hybridization to ribosomal RNA is observed, increase the prehybridization, hybridization, and washing temperatures to 68 °C. Beware that this is often caused by the oven temperature calibration drifting such that the oven is not actually at the set temperature of 65 °C. Leave a beaker of water overnight in the oven and check the temperature with a trustworthy thermometer to be sure. In contrast, if the probe is unavoidably AT rich (such as the probe used in Fig.2d) and a very low signal is obtained, reduce the prehybridization, hybridization and washing temperature to 60 °C and hybridize again with the same probe. Further temperature optimization is unlikely to improve the signal.
?. [Hybridization of RNA Probes]
To make the probe, mix the following reagents in the given order at  in a 1.5 mL screw cap tube: (the absolute amount does not seem to be critical). 3000 Ci/mMol [α-32P] rUTP
65 Room temperature
[gel purified template]
[100 μM rUTP]
[milliQ H2O]
[10 mM rCTP/rGTP/rATP]
[100 mM DTT]
[10 mg/mL BSA]
[5× transcription buffer]
2.5 µl
[T7 RNA polymerase]
Some cold UTP is important otherwise the in vitro transcription is inefficient. However, more cold UTP means that the radioactivity per probe molecule is lower, reducing the signal. This amount is a compromise, try not to change unless absolutely necessary.
It is critical that the polymerase is added after the radioactive rUTP, then mixed well.
?. [Hybridization of RNA Probes]
Mix well and incubate – at .
37 °C
In our hands   or more of labeling is fine.
?. [Hybridization of RNA 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 RNA 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 RNA Probes]
Dilute the probe with water to  and then pipette carefully on to the center of the sepharose matrix in the column.
50 µl
?. [Hybridization of RNA Probes]
Centrifuge at .
Centrifuge: 1000 34, 4 min
?. [Hybridization of RNA 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 RNA Probes]
Add the probe to the hybridization bottle while vertical so that it falls directly into the hybridization buffer, and continue to rotate at .
65 °C
?. [Hybridization of RNA 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 RNA Probes]
Pour ~ in the bottle, close tightly and shake gently then pour off the wash solution. (1/2)
[6× SSC]
The washes with 6× SSC are the low stringency washes. After the probe is poured off, the blot is washed repeatedly with low stringency wash buffer (high salt) to remove unbound probe.
?. [Hybridization of RNA Probes]
Pour ~ in the bottle, close tightly and shake gently then pour off the wash solution. (2/2)
[6× SSC]
?. [Hybridization of RNA Probes]
Add , return the bottle to the hybridization oven and rotate at  for at least . Make sure the bottle is balanced by another also containing .
[6× SSC]
65 °C
[liquid]
?. [Hybridization of RNA Probes]
Pour off the 6× SSC and add , return to hybridization oven and rotate at  for . (1/2)
[prewarmed (65 °C) 0.1× SSC, 0.1% SDS]
65 °C
The washes with 0.1× SSC 0.1% SDS are the high stringency washes. The blot is washed 2–3 times with high stringency wash buffer (low salt) at high temperature to remove nonspecifically bound probe.
?. [Hybridization of RNA Probes]
Pour off the 6× SSC and add , return to hybridization oven and rotate at  for . (2/2)
[prewarmed (65 °C) 0.1× SSC, 0.1% SDS]
65 °C
?. [Hybridization of RNA Probes]
Pour off the remaining buffer, and use tweezers to pull the membrane out of the bottle onto a paper towel.
To clean the bottle, simply rinse with water and give it a wipe with a paper towel to remove residual counts. The lids are harder to clean: run water into the lid (hopefully this will dislodge the rubber seal), and wipe inside with a paper towel. Do not attempt to prise the seal out as this can damage the seal, leading to leaks during future hybridization. It is often not possible to completely decontaminate hybridization bottle lids, so we always treat them as if contaminated. With careful handling, the bottles, lids, and seals last for many years.
?. [Hybridization of RNA Probes]
Pat lightly to leave semidry. It is wise to do this behind a Perspex shield as membranes sometimes carry a substantial amount of radioactivity.
?. [Hybridization of RNA Probes]
Wrap the membrane in Saran Wrap.
?. [Hybridization of RNA Probes]
Check the signal with a Geiger counter to obtain an estimated exposure time. Table 1Storage phosphor screen exposure time guidelines  ABC1Counts per secondExposure time (h)Quality2&lt;5024Able to see and quantify but poor quality336More sensitive and good quality450–4002Able to see and quantify but poor quality548Good quality696Over exposed (will impede quantification)7&gt;4002Good quality
If the exposure time needs to be adjusted, the phosphor screens can be blanked and reexposed. For very intense signals (thousands of counts per second) the phosphor screen may need to be exposed to natural UV light for several days to fully blank the signal (i.e., leave it on the windowsill!).
ABC1Counts per secondExposure time (h)Quality2&lt;5024Able to see and quantify but poor quality336More sensitive and good quality450–4002Able to see and quantify but poor quality548Good quality696Over exposed (will impede quantification)7&gt;4002Good quality
?. [Hybridization of RNA Probes]
Expose the membrane to a storage phosphor screen in a cassette. The screen should be blanked just before use as it will expose slowly with time.