TITLE: Northern Blot Protocol
AUTHORS: Sibylle Mitschka, Christine Mayr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Northern blot is a hybridization technique to visualize and quantify RNAs from cells or tissues using a radio-labeled probe. mRNAs only constitute a small fraction of total RNA in cells. Through enrichment of poly(A)-tailed mRNAs, this protocol provides increased sensitivity for the detection of lowly expressed mRNAs. In addition, RNA denaturation is achieved using glyoxal (in contrast to formaldehyde-based methods), resulting in sharper bands. We have used this protocol extensively to detect and quantify the expression of different 3'UTR isoforms originating from the same gene </span><span style = "vertical-align:super;">1-5</span><span style = "vertical-align:super;">.</span></div><div class = "text-block"><span style = "vertical-align:super;">1 </span><span>Mayr, C. and D. P. Bartel (2009). Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation  activates oncogenes in cancer cells. Cell 138(4): 673-684. </span></div><div class = "text-block"><span style = "vertical-align:super;">2 </span><span>Lianoglou, S., V. Garg, J. L. Yang, C. S. Leslie and C. Mayr (2013). Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression. Genes Dev 27(21): 2380-2396.</span></div><div class = "text-block"><span style = "vertical-align:super;">3</span><span> Berkovits, B. D. and C. Mayr (2015). Alternative 3'UTRs act as scaffolds to regulate membrane protein localization. Nature 522(7556): 363-367.</span></div><div class = "text-block"><span style = "vertical-align:super;">4</span><span> Lee, S. H. and C. Mayr (2019). "Gain of Additional BIRC3 Protein Functions through 3'-UTR-Mediated Protein Complex Formation. Mol Cell 74(4): 701-712 e709.</span></div><div class = "text-block"><span style = "vertical-align:super;">5</span><span> Mitschka, S. and C. Mayr (2020). Endogenous p53 expression in human and mouse is not regulated by its 3'UTR. bioRxiv: 2020.2011.2023.394197.</span></div></div>

[STEPS]
?. [Total RNA extraction]
Thaw TRIzol samples at RT.
?. [Chloroform extraction]
add    per tube
200 µl
?. [Chloroform extraction]
Mix tubes vigorously for
?. [Chloroform extraction]
Allow samples to sit for  at
0 Room temperature
?. [Chloroform extraction]
Centrifuge samples at
Centrifuge: 20000 34, 20 min, 4 10
?. [Chloroform extraction]
Transfer clear upper phase into a new tube (about  to  )
400 µl
500 µl
?. [Chloroform extraction]
Transfer clear upper phase into a new tube (about  to  )Add
400 µl
500 µl
500 µl
?. [Chloroform extraction]
Add
500 µl
?. [Chloroform extraction]
Vortex samples and store at   for   to
-20 °C
?. 
?. [Chloroform extraction]
Centrifuge samples at
Centrifuge: 20000 34, 20 min, 4 10
?. [Chloroform extraction]
Pour off the supernatant (the pellet should be stuck to the wall of the tube).
?. [Chloroform extraction]
Add  to each tube.
1 mL
?. [Chloroform extraction]
Centrifuge
Centrifuge: 20000 34, 10 min, 4 10
?. [Chloroform extraction]
Pour off the supernatant and remove all liquid with a pipet tip.
?. [Chloroform extraction]
?. [Chloroform extraction]
?. [Total RNA extraction]
Add 200 μl chloroform per sample and mix vigorously for 15 sec. Then allow to sit for 3 min at RT.
?. [Total RNA extraction]
Centrifuge 20,000 g/20 min/RT.
?. [Total RNA extraction]
Transfer clear upper phase to a new tube and add 500 μl isopropanol. Vortex briefly.
?. [Total RNA extraction]
To facilitate RNA precipitation, incubate samples for 2 h to overnight at 80 °C.
?. [Total RNA extraction]
Centrifuge 20,000 g/20 min/4 °C.
?. [Total RNA extraction]
Pour off the supernatant and add 1 ml 70% ethanol to each sample.
?. [Total RNA extraction]
When RNA pellet appears translucent, add between 10 μl and 60 μl RNase-free water, depending on the size of the pellet. Incubate for 30 min on ice and resuspend the RNA by repeated pipetting.
?. [Total RNA extraction]
Pour off the supernatant and carefully remove remaining liquid with a small pipet tip. Let the RNA pellet air-dry for 5 min.
?. [Total RNA extraction]
Centrifuge 20,000 g/10 min/4 °C.
?. [Total RNA extraction]
Make a 1:10 dilution of the RNA solution  (e.g 0.5 μl RNA plus 4.5 μl water) and measure the RNA concentration using a NanoDrop spectrophotometer. The RNA solution can be stored at -80 °C but repeated freeze-thaw cycles should be avoided.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Transfer up to 180 μg of total RNA into a new tube and adjust volume to 250 μl with water.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Vortex Oligotex bead suspension (Qiagen kit) to homogenize the solution and add 15 μl to each tube.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Add 250 μl buffer OBB (Qiagen kit) and mix.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Incubate samples in a 70 °C heat block for 5 min.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Then place samples in the 25 °C water bath for 15 min.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Centrifuge 20,000 g/2 min/RT.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Pipet off the supernatant and resuspend beads in 400 μl OW2 (Qiagen kit).
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Place a spin column into a 1.5 ml tube and add the Oligotex bead mixture. Centrifuge 20,000 g/1 min/RT.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Transfer the spin column to a new tube (the flow-through is discarded) and add another 400 μl OW2. Centrifuge  20,000 g/1 min/RT .
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Transfer column to a new labeled tube and add 100 μl pre-warmed buffer OEB (Qiagen kit). Resuspend beads by pipetting and place samples in a 70 °C heat block for 1 min.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Centrifuge 20,000 g/1 min/RT .
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Repeat steps 21-22 to collect another 100 μl of eluate in the tube (total volume 200 μl).
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Add 20 μl 3 M sodium acetate solution, 1 μl GlycoBlue and 550 μl 100 % Ethanol. Mix and incubate samples for 2 h to overnight at -20 °C.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Pour off the supernatant and add 1 mL 70% ethanol to each sample.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Centrifuge 20,000 g/10 min/4 °C.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Pour off the supernatant and carefully remove remaining liquid with a small pipet tip. Let the RNA pellet air-dry for 3 min.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
When RNA pellet appears translucent, add 3 μl RNase-free water. Resuspend by incubating on ice and repeated pipetting.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Make a 1:10 dilution of the RNA solution (e.g. 0.3 μl RNA plus 2.7 μl water) and measure its concentration using a NanoDrop spectrophotometer. It is best to use the mRNA immediately but it can be stored at -80 °C.
?. [Final sample preparation and running the gel]
For each sample, transfer an equal amount of mRNA to a new tube (e.g. 2 μg) and adjust volume to 3 μl with water. Keep samples on ice. Prepare an additional tube for the marker RNA containing 3 μl RiboRuler and process it in parallel with the other samples.
?. [Final sample preparation and running the gel]
Add 10 μl  glyoxal mix to each tube and incubate samples in a 55 °C heat block for 1 h.
?. [Final sample preparation and running the gel]
In the meantime, prepare a 1% agarose gel by mixing 1 g GTG Seakam Agarose with 100 mL 1x TAE buffer (RNase-free). Bring the agarose to boil in a microwave (about 2 min). Let the melted agarose cool a bit and pour the mixture into a clean agarose gel tray. Make sure that the surface is very even and add a thin gel comb  Let the gel solidify at RT.
?. [Final sample preparation and running the gel]
After one hour, briefly spin down RNA samples and add 2 μl RNA-loading dye to each tube.
?. [Final sample preparation and running the gel]
Load entire sample volume of 15 μl into wells of prepared agarose gel.
?. [Final sample preparation and running the gel]
Run the agarose gel in 1x BPTE buffer at 4 °C for 1.5 h at 120 V. The darker dye front should have migrated approximately 8 cm (3.1 in) from the well.
?. [RNA blotting]
After the run is complete, take a picture of the gel using the GelDoc imaging system. In intact RNA samples, the ribosomal RNA bands should be sharply defined with 28S/18S rRNA intensity ratios of about 2:1.
?. [RNA blotting]
Assemble the blot according to the manufacturer’s instructions. Be sure to avoid any air bubbles between membrane and gel. Fill the outer reservoir of the chamber with 20x SSC and transfer overnight. Do not put any weights on the blot stack.
?. [RNA blotting]
Put the membrane into a Pyrex dish filled with 200 mL 2x SSC and shake it on an orbital shaker for 5 min.
?. [RNA blotting]
Transfer the membrane into a new Pyrex dish containing 200 mL 20 mM Tris pH 8.0 and incubate for 25 min. This step reverses the glyoxal cross-linking reaction.
?. [RNA blotting]
Dry the membrane on a piece of filter paper.
?. [RNA blotting]
Crosslink the RNA to the membrane by exposing it to 1,000 J in a UV crosslinker.
?. [RNA blotting]
In a clean Pyrex dish, soak the gel, membrane and some of the filter papers in 20x SSC for 5 min.
?. [RNA blotting]
Mark the gel orientation on the membrane with a pencil. The blot can be cut using scissors to enable simultaneous staining with different DNA probes.
?. [Pre-hybridization and probe preparation]
Place blot in a glass cylinder with the top side facing inside and add  17 mL of the pre-warmed ULTRA-Hyb buffer to each hybridization tube. Close the hybridization tube securely and make sure that the gasket is inside the cap. Roll the closed tube with the buffer inside until the blot sticks to the inside of the tube. Avoid air bubbles between glass and membrane. Use long forceps to adjust the membrane if necessary.
?. [Pre-hybridization and probe preparation]
Place the hybridization tube into the 42 °C oven and rotate on a low speed setting. Pre-hybridize the membrane for at least 1 h.
?. [DNA Probe labeling]
Purify the product and generate a single-stranded radio-labeled probe in a single primer PCR reaction (step 54). Alternatively, the second step of probe labeling can be carried out using a commercial kit (Prime-A-Gene Labeling System from Promega, U1100).
?. [DNA Probe labeling]
Amplify the target region from cDNA with gene-specific primers using standard PCR procedures. Run the whole reaction on an agarose gel and perform gel extraction using  the QIAquick Gel Extraction kit according to the manufacturer's instructions. Measure the concentration of the template DNA using the NanoDrop.
?. [DNA Probe labeling]
Set up the labeling reaction (single primer PCR) behind a plexiglass shield:2 μl 10x Taq buffer 2 μl 10x dNTP mix without dATP (i.e. dTTP, dCTP, dGTP, 2 mM each)1 μl reverse primer (10 μM)30 ng purified DNA template from step 535 μl [α-32P] dATP 1 μl Taq DNA polymeraseadd sterile water to a total volume of 20 μl
?. [DNA Probe labeling]
Run the following program in a PCR thermocycler: Initial denaturation 95 °C for 2 mindenature at 95 °C for 30 secAnneal at 45 °C for 2 minAnneal at 72°C for 30 secRepeat steps 2-4 35 times
?. [DNA Probe labeling]
Add 5 μl of 0.2 M EDTA to the PCR reaction (the total volume is now 25 μl ) and heat to 95 °C for 5 min. Afterwards, place the probe immediately on ice.
?. [DNA Probe labeling]
Add the entire probe volume to the hybridization tube containing the blot membrane.Continue to incubate the blot rotating at 42 °C overnight.
?. [Washing the blots and exposure]
This protocol describes the use of the phosphorimaging system for signal detection, as it provides a greater dynamic range and increased sensitivity. However, X-ray autoradiographic films can be used as an alternative (step 64).
?. [Washing the blots and exposure]
The next day, pour off the UltraHyb + probe into a falcon tube (if you are going to re-use the probe), or into the liquid radioactive waste container. If you are saving the buffer/probe in a falcon tube, transfer it carefully and store the falcon tube in a radiation-resistent plexiglass box at 4 °C. Use within a week.
?. [Washing the blots and exposure]
Add 30 mL of Wash Buffer 1 to the hybridization tube and wash for 10 min in the hybridization oven, rotating at fairly high speed.
?. [Washing the blots and exposure]
Pour off Wash Buffer 1 and repeat step 60.
?. [Washing the blots and exposure]
Pour off Wash Buffer 1 and add 30 mL Wash Buffer 2. Wash in Wash Buffer 2 for 20 min.
?. [Washing the blots and exposure]
Pour off Wash Buffer 2. Use forceps to take the blots out of the tubes and place them into a clean plastic sleeve for exposure.
?. [Washing the blots and exposure]
For a first estimate of the signal, expose the blot for 1 h in a cassette (put down blots in their sleeves, then put a storage phosphor screen over the blot and close cassette, noting the date and time). After an hour, take a picture of the blot in the Phosphorimager and re-expose for the desired time (up to several days).
?. [Stripping of blot membranes]
Warm 250 mL 0.5% SDS (50 ml 10% SDS in 1 L water) to a boil in a microwave. For 250 mL, this takes about 2:30 minutes. Use a large glass beaker as the solution has very low surface tension leading to overflow when boiling.
?. [Stripping of blot membranes]
Allow the 0.5% SDS solution to cool for 20 sec until all the bubbles have settled.
?. [Stripping of blot membranes]
Put the blot into a Pyrex dish on the shaker and carefully add on the 0.5% SDS (not directly on the blot).
?. [Stripping of blot membranes]
Shake for at least 20 minutes at RT.
?. [Stripping of blot membranes]
Pour off the 0.5% SDS.
?. [Stripping of blot membranes]
Repeat steps 1-5 for at least another 2 times.More washes remove more of the radio-labeled probe but also diminish the remaining signal on the blot.
?. [Stripping of blot membranes]
Remove the blot from the dish and let it dry for 15 min on dry Whatman paper. Store the blot in a plastic sleeves or prepare for hybridization. Alternatively, expose the blot for 1 hour to see how well the stripping worked.
?. [Enrichment of poly(A) mRNAs with Oligotex beads]
Before starting, set a heat block to 70 °C and the water bath to 25 °C. Tubes containing the required amount of buffer OEB are pre-warmed in the 70 °C heat block.Poly(A) mRNA enrichment using Oligotex beads should be performed in batches of not more than 8 samples.
?. [Final sample preparation and running the gel]
Make required buffers and reagents ahead of time. Thaw the glyoxal mix and RiboRuler on ice. The glyoxal mix should be handled with care because it contains ethidium bromide. Set a heat block to 55 °C. Clean all equipment with water and RNase Away surface decontaminant.
?. [Pre-hybridization and probe preparation]
Set the hybridization oven to 42 °C and pre-warm the ULTRA-Hyb buffer in the oven for an hour or until the entire contents of the bottle are liquid and warm.
?. [Total RNA extraction]
This protocol requires samples that have previously been homogenized in TRIzol. Samples can be stored at -80 °C for several months.High quality RNA is paramount for the success of this procedure. To avoid RNase contamination, only use filter tips when handling RNA-containing samples and clean equipment with water and surface decontaminant.
?. [RNA blotting]
The next morning, record another picture of the agarose gel. There should be a substantial and even loss of RNA signal in the gel relative to the pre-blot picture from step 39.
?. [RNA blotting]
Afterwards, place blot in the hybridization oven at 80 °C for 30 min.
?. [Stripping of blot membranes]
Blot membranes can be stripped for re-probing several times with the protocol described below.  It is strongly suggested to begin by probing the blot against the mRNA with the lowest expression, as each stripping reduces the signal strength. Furthermore, very strong hybridization signal cannot be completely removed by stripping. This can cause specific "shadow" band to appear in subsequent exposures. Always note the bands of all prior hybridizations. With the decay of the radioactivity, the probe signal is also disappearing naturally over time (phosphor 32 has a half-life of about 14 days). If stored at -20 °C, the membrane can be re-probed over several months.