TITLE: RNA Electrophoresis in Agarose Gels
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 Although superseded by qPCR and sequencing methods for routine mRNA quantification, northern blotting is the method of choice when complex mixtures of overlapping species are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]
?. [RNA Electrophoresis in Agarose Gels]
Note that the method described below is for a 15 × 15 cm gel.
?. [RNA Electrophoresis in Agarose Gels]
Add ethidium bromide to an aliquot of glyoxal mix to a final concentration of .
?. [RNA Electrophoresis in Agarose Gels]
Mix  with  (can use less of RNA sample), then incubate at for .
[glyoxal/EtBr]
[RNA sample]
55 °C
The ratio of glyoxal mix to RNA should be at least 5:1, but varying the ratio does not alter the result so samples do not all need to be at the same concentration. Glyoxylation at 55 °C can be left for longer than an hour if the gel is not ready to be loaded. Do not take the samples out and let them sit at room temperature. In our experience, if samples are left for too long at room temperature migration is compromised.
?. [RNA Electrophoresis in Agarose Gels]
Mix  with , add  and incubate at  along with the RNA samples.
[DNA ladder]
[glyoxal mix]
[10 mg/mL ethidium bromide]
55 °C
?. [RNA Electrophoresis in Agarose Gels]
Make a 15 × 15 cm 1.2% agarose gel by mixing  with .
[agarose]
[1× BPTE]
Making a thicker gel to be able to fit more sample in is not a good idea as RNA transfer can be compromised. Also, make sure the agarose is fully melted as agarose lumps impair the transfer.
?. [RNA Electrophoresis in Agarose Gels]
Fill the tank with 1× BPTE.
?. [RNA Electrophoresis in Agarose Gels]
Centrifuge the samples briefly and load on the gel. If a spare lane is available, add  to track migration.
[bromophenol blue loading dye]
?. [RNA Electrophoresis in Agarose Gels]
Run the gel at 150 V until bromophenol blue is ~ from the bottom (about ). This keeps all RNA on the gel, or migrate further if required. The voltage for this electrophoresis should be no higher than 6 V/cm, based on the distance between the electrodes.
?. [RNA Electrophoresis in Agarose Gels]
Acquire a digital image of the ethidium staining pattern using a trans-illuminator or fluorescence imager to allow quantification and assessment of molecular weight based on the marker.
?. [RNA Electrophoresis in Agarose Gels]
Wash the gel  with .
[NaOH]
It is critical that the NaOH wash is done at the specified concentration and length of time for the RNA to break into the right size fragments. This is to ensure good transfer and good hybridization of the probe.
?. [RNA Electrophoresis in Agarose Gels]
Rinse the gel with milliQ water.
?. [RNA Electrophoresis in Agarose Gels]
Wash twice with neutralization solution for , all on a shaker. (Wash 1/2)
The neutralization solution washes can be longer. Up to 1.5 h works fine in our hands.
?. [RNA Electrophoresis in Agarose Gels]
Wash twice with neutralization solution for , all on a shaker. (Wash 2/2)
The neutralization solution washes can be longer. Up to 1.5 h works fine in our hands.