Detection and quantitation of NAD+- and NADH-capped RNA in vivo: hybridization with a radiolabeled oligodeoxyribonucleotide probe

I: DNAzyme treatments of RNA samples

- For a detailed explanation of DNA enzymes (DNAzymes) for cutting of RNAs in vitro please see Joyce. Methods in Enzymology (2001) and Santoro and Joyce. PNAS (1997). These references outline how to design a 10-23 DNAzyme for site specific cutting of a target RNA.

- DNAzyme cleavage of target RNAs ensures that target RNAs have uniform 3′-ends, which allows differentiation of different 5′-end structures by gel electrophoresis.

DNAzyme treatments are done in NEB Buffer 2 (10 mM Tris pH7.9 [pH 8 works too if making from scratch], 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT)

1.  Add Buffer, RNA and 10 uM DNAzymes into an appropriate tube (PCR tube if using PCR machine; 1.7 mL Eppendorf tube if using a heat block/water bath). I normally will treat 50 to 100 µg of RNA (based on the inaccurate nanodrop reading above) in one 50 µl rxn.

- Up to 5 DNAzymes in one reaction has been done successfully. It is possible that more would be possible, but we have not tried yet.

- NudC (E. coli decapping enzyme) treatment is used as a control to identified capped species on the blot

2a. Incubate rxns for 1 hr at 37^oC

2b. For most applications (with properly designed DNAzyme sequences), the one hour at 37^oC should be sufficient. If you are trying to cut RNA with a high amount of secondary structure, the protocol needs to be modified slightly:

1) Use modified NEB buffer 2 that does not contain MgCl₂ in the preparation of the rxns

2) On a PCR machine heat rxns containing DNAzymes, RNA and no Mg buffer to 85^oC for 5 minutes to disrupt RNA secondary structure

3) Perform a slow ramp to 37^oC (approx. 0.1^oC per second) to anneal DNAzymes to target RNAs

4) Once at 37^oC, Add MgCl₂ to 10 mM (5 µl of 50 mM stock) and then add NudC enzyme (it will denature during the heating step otherwise). Incubate for 1 hr as in 2a.

3. Stop reactions by adding 100 µl ice cold 1.5x STOP solution (50 mM Tris pH 8.0, 20 mM EDTA, 0.1 µg/mL glycogen)

4. Add 450 ul ice cold EtOH to precipitate RNAs and incubate at least one hour at -20^oC minimum. Overnight is preferable.

5. Spin RNA samples for 30 minutes at 13k rpm at 4^oC

6. Remove supernatant. Spin again 30 seconds and collect the remaining liquid.

7. Dry samples on the benchtop for 5 minutes.

8. Resuspend RNAs in 1x RNA loading dye (50 mM Tris pH 8.0, 0.2% SDS, 25 mM EDTA, 90% deionized formamide, xylene cyanol, bromophenol blue; store loading dye at -20^oC until use). I will usually resuspend my RNA in 1 µl RNA loading dye per 2.5 µg of RNA (again based on the nanodrop reading).

- For most RNA applications you would normally boil samples before loading on a gel. DO NOT boil NAD⁺/NADH-capped RNA samples as the NAD is heat labile and will degrade. Keep RNA samples in loading buffer at -20 or -80^oC until ready for use. Keep samples on ice while manipulating them/loading the gel.

II: Electrophoresis and Transfer

- The methods used below are adapted from the previous Nickel’s lab protocol for northern blotting using EDC crosslinking, but here, using UV crosslinking instead. For reference: Goldman et al. eLife (2015).

1a.  Cast a 0.4mm denaturing polyacrylamide gel with 0.2% acrylophenylboronic acid (0.14g for 70 ml gel solution) using Bio-Rad Sequagen GT gel system (38 x 30 cm format) and the National Diagnostics Urea gel system buffers.

- Lower percentage gels will be easier to manipulate, but up to 22.5% work fine for this system. Note that for NCIN band shift Northerns (for RNA products in the 30-100nt range) 8% or 10% gels work best.

- Be sure to siliconize one plate of the sequencing gel rig so that you ensure the gel sticks to the opposite plate. For the Bio-Rad system, siliconize the Upper-buffer reservoir plate using Rain-X spray.

2.  Prepare 0.5 L of 0.5x TBE (store at 4^oC) and thaw Denhardt’s solution for later use (NOTE: Using TBE for the transfer will not affect the separation of capped species in the gel as it is after the gel running step).

3.  Pre-run the gel for at least 15 minutes prior to loading using 1x TBE as running buffer.

4.  Load the samples and run gel. For strong signal from COX2 blots from yeast culture using JB555 COX2 Northern probe, 5 µg of RNA for YPEG culture samples (Respiratory conditions) and 10-20 µg from YPD cultures gives sufficient signal. The RNA loading amount will be determined by the relative abundance of the transcript of interest. In addition to samples, load an appropriate amount of 32P-labeled Decade RNA marker (Thermo Fisher). The amount of marker to use will depend on how recently it was made and the relative final signal from experimental samples. 

- Make sure to run a lane of RNA loading dye with amaranth near your samples on the gel!!! On 0.2 mm sequencing gel amaranth runs at approximately the same place as free nucleotide and will allow you to better estimate where your desired bands are located.    

- Bands of interest (20-100 nt) run roughly near the bromophenol blue dye band. It's okay to let the amaranth band overrun the desired final blot size. You will cut the gel to the size of your blot for transfer.

- Bear in mind that the maximum size of gel that can be transferred on a Bio-Rad Transblot SD semi-dry apparatus is 24 x 17 cm.

- For NCIN band shift you should run the gel 1 hr at least (until there is approximately 10 cm between the BPB and XC dye bands). If you wish to visualize the whole range of RNAs, run the gel a shorter time such that you can blot the whole region from the wells to the Amaranth dye.

While the gel is running: 

5. Cut filter paper and nylon membrane to appropriate size. Cut 9 pieces of filter paper to the same size as the membrane and a 10^th piece to the size of sequencing gel you are running.

- Handle the membrane with great care. Wear gloves. Any creases, folds or dings will collect probe and result in lines, dots or background on your blot image.

- It is also very important to keep track of the orientation of the gel and the membrane. For example, cut off the upper left-hand corner of the membrane and cut a small vertical slit (or poke a needle hole) in the lower right hand corner. This is the sense orientation and will be how you will expose the membrane for imaging. 

6.  Also prepare prehybridization/hybridization (prehyb/hyb) solution and prepare wash solutions for the next day. Turn on the hybridization oven at 50°C and pre-warm hyb bottles (important to minimize risk of leakage).

- Select a hyb bottle large enough that the membrane can unroll completely without any overlapping edges. Overlapping edges don’t get the same exposure to solutions and often result in “wash out” where the exposure in the edges will be faint.

Prehyb/Hyb solution

For 15 cm hyb bottles, halve all the volumes: 25 mL each for prehyb and hyb. 

For 30 cm bottles, make 2 x 50 mL aliquots.

Incubate at 55°C until equilibrated.  At that time add 200 ul per 50 mL of Sheared Salmon Sperm DNA solution (10 mg/mL) that has been denatured at 95°C for at least 5 minutes. 

Wash solutions

Prepare wash solutions the night before use and place at 55°C until needed.  300mm bottles refer to both the 30 x 300mm and 70 x 300mm bottles.

Non-Stringent Wash (NSW)

Stringent Wash (SW)

7.  When the run is finished, stop the gel and dispose of all running buffers using the aspirator in the hot lab.

8.  Lay out a piece of bench paper and dismantle the apparatus and separate the plates. The gel should stick to the front (non-siliconized) plate.

- These steps are messy. Work on bench paper and change gloves often. Remember that the Decade marker is radioactive so work behind a shield as much as possible. Some steps may not be easily performed behind a shield. If you elect not to use the shield, work efficiently, and maintain distance as much as possible.

9.  Transfer the gel from the glass plate to large piece of filter paper.

- This can be a little tricky. The gel is strong but brittle and prone to tears. Also the addition of the acrylophenylboronic acid may give the gel somewhat of a gummy texture that can be hard to manipulate. Be very patient and use a light touch.

10.  Cover the gel with plastic wrap making sure to not have wrinkles.

11.  Using a metal straight-edge and a new razor blade, crop the gel/filter paper to the same size as your cut blot. Be sure to remove the lane dividers at the top, unused gel on the sides and below the amaranth dye at the bottom (for visualizing the full range of RNA). For the NCIN blots it is worth cutting region of interest from your gel (between the BPB and XC bands on the gel usually).

- When cropping the top of the gel, remove the lane dividers but do go below them (you may lose sample). It is fine if short stumps of the dividers remain attached. Dividers are removed only to reduce the risk of tearing the gel during handling.

- The amaranth dye migrates at about 2-nt, so it is below the minimum detectable size by northern (around 10-nt). If the gel is too tall, you can crop from the amaranth end (the bottom) without losing sample.

12.  Remove excess gel and plastic wrap.

13.  Fill a large baking dish with 0.5 l of the chilled 0.5x TBE.

14.  Soak the pieces of filter paper in the 0.5x TBE until they are fully wet (~30 sec).

15. Place a piece of plastic wrap on the bench top and carefully place a stack of 4 pieces of the wet filter paper.

16.  Gently soak the piece of filter paper with your gel on it in the 0.5x TBE and place it on the filter paper stack. Then carefully remove the plastic wrap from the cropped gel.

17.  Soak the cut membrane in the 0.5x TBE (15-30 seconds).

18.  Lift the membrane by opposite corners and lay it on the gel such that the cut corner is at the upper right (near the xylene cyanol dye) and the slitted corner is at the lower left.

- Once the membrane is in contact with the gel, some transfer will commence immediately so try to get the position of the membrane correct on the first try.

19.  Take the remaining 5 pieces of soaked filter paper and carefully lay them on top of the membrane. 

- This is the lower half of the transfer stack. The Bio-Rad transblot cell transfers downward (bottom plate carries positive charge).

20.  Using a 50 ml conical, gently roll across the stack to remove air bubbles between any of the layers (especially between the membrane and gel). RNAs will not transfer to the membrane through air bubbles.

21.  Pick up the stack and invert it (membrane side down) and place on the transfer apparatus

22.  Roll air bubbles out of the stack with your 50 ml conical.

- The goal here is to remove air bubbles, not to wring the stack dry so don't press down too hard.

23.  Use paper towel to mop up excess buffer squeezed out of the stack.

24.  Optional: Add 1 ml of additional buffer to the top of the stack.

25.  Secure the lid and the safety lid.

26.  Transfer the blot at 25V constant for 25 minutes.

- If everything went well the current will start very high (1-2 A; ideal) and rapidly fall to 1-200 mA by the end of the run. The semidry apparatus should never be run at greater than 25V under any circumstances. Mind the polarity of the power cords as the apparatus should not be operated in the reverse direction.

27.  While the transfer is running, prepare the probe labeling reaction and thaw/boil your salmon sperm DNA to add to the Prehyb/hyb solution.

- Always use the freshest available γ-32P-ATP in the probe labeling rxn. Northern blot signal with small oligo probes tend to be low and fresh label ensures the best possible final image.

Probe end-labeling:

*LNA oligos can be resuspended in RNAse free TE (such as IDTE) or 0.1X Qiagen EB.  DEPC water is also okay.

Incubate 37°C for one to two hours. Heat inactivate 95°C for 5 minutes. Clean up with G-25 microspin column (Sigma-Spin columns or equivalent). Optional: Quantify via scintillation. If activity is less than 600,000 cpm/µl final signal on the blot will be very low. It should routinely be possible to obtain activities in excess of 10⁶ cpm/ µl

28.  Cut a piece of filter paper slightly larger than the membrane, wet it in the 0.5x TBE and lay it on a piece of folded over plastic wrap.

29.  Dismantle the transfer apparatus and stack. The side of the membrane that is facing your gel in the stack is now the RNA side of the membrane.

30.  Lay the membrane RNA side up on the new piece of filter paper (that has been soaked in the 0.5x TBE solution from earlier) and put it in the Fisher UV crosslinker. Turn the crosslinker on. Hit the Optimal crosslink button and then hit start (this will take about 30 seconds). Once it is done rotate the blot 180 degrees in the crosslinker and hit start again. Your RNA is now crosslinked to your blot.

31.  Cut a piece of hybridization mesh slightly larger than your membrane. Lay it on a piece of filter paper sitting on piece of plastic wrap.

- The filter paper absorbs excess liquid and makes it easier to reposition the membrane if need be.

32.  Transfer the membrane, RNA side up, onto the mesh. Roll the mesh up starting at the right side. Place it into a Hyb bottle.

33.  Add prehyb solution. Close the hyb bottle securely but don’t over-tighten. Place in hyb oven.

34.  Incubate 50°C for 30-60 min.

35.  Decant prehyb solution into radioactive waste.

- Drips down the outside of the bottle are a major source of radioactive contamination. To avoid this problem, keep a stack of small cut paper towels on hand and catch the drips before they run into the threads on the outside of the hyb bottle.

36.  Add entire labeled probe into the other aliquot of prehyb solution. Mix well. This is the hybridization solution.

37.  Add hybridization solution to bottle, close and incubate overnight. Ideally this should go for at least 12 hours with 16-18 hours being closer to ideal.

IV: Washing and imaging

- Washes for Northern blotting are done in two stages: non-stringent and stringent. Non-stringent wash contains a lot of salts and detergent which help stabilize probe/target interactions. Stringent wash is relatively dilute and will destabilize poorly matched probe/target interactions. Pure water would be the most stringent wash possible.

38.  Decant the hyb solution into hot waste. This will be very radioactive!

39.  Wash with non-stringent solution at 50°C 4x 15 minutes. Use 50 ml per wash for 30 cm hyb bottles.

- Add the wash solution carefully and avoid pipetting it directly against the blot if possible. It seems to be possible to wash out areas of the blot by exposing the too forcefully to wash solution.

40.  Wash once with 80 ml stringent solution for 5 minutes at 50°C.

- At this time, take a large phosphor screen and set it on the image eraser. 

41.  Decant final wash solution into a clean baking dish.

42.  Remove membrane from bottle by grasping the hyb mesh with forceps and pulling gently. Be careful not to scratch the inside of the hyb bottle in the process.

43.  Place the membrane into the baking dish with the wash solution, unroll it and remove the hyb mesh (discard as hot waste)

44.  Place a piece of filter paper slightly larger than the membrane onto a piece of plastic wrap on the bench.  Place another clean piece of plastic wrap sufficient to wrap the blot on the bench next to it.

- You will take the blot from wash solution, remove excess liquid and then wrap it for exposure.

45.  Carefully lift the membrane out of the baking dish and lay it face down onto the filter paper.  Let it sit for a few seconds.  Apply another piece of filter paper on top to remove liquid from the back.

- The goal is to remove residual stringent wash solution which may stil be radioactive and can show up as background

46.  Transfer the blot, RNA side down, to the clean plastic wrap. Wrap it carefully to avoid bubbles.

47.  Tape the wrapped membrane into phosphor cassette, retrieve the screen from the eraser and lock it onto the cassette.  Expose as needed and image using the phosphorimager.

- It will usually be possible to see if you blot worked after 1 hour of exposure, but 24+ hours are usually needed for a quality image.

Solutions:

DEPC Treated Water (use for all subsequent solutions)

      1 L ddH₂O

      1 ml DEPC

Stir for at least 1 hr up to overnight at room temp. Autoclave 60 min to decompose residual DEPC.

50X Denhardt's Solution 

      1% BSA fraction V (high purity)

      1% Ficoll 400

      1% polyvinylpyrrolidone (PVP)

Add water and stir. Can be very slow to dissolve, but is made much faster if the water is pre-warmed to 55°C before adding dry components. Filter sterilize.  Make 40 mL aliquots in conical tubes and store at -20°C. 

20% SDS

      200 g SDS

      water to 1 L

Gentle heating will help dissolve the SDS faster. Wear a dust mask as SDS is very irritating to the lungs. Filter sterilize.

20X SSC

      3 M Sodium chloride

      0.3 M Sodium citrate

To make one liter add 175.3g NaCl and 88.2g Sodium citrate (dihydrate).  Bring to 800 mL with water.  Adjust to pH 7 (a few drops of HCl).  Bring to 1 l and filter.

1M Na₂HPO₄ (pH 7.2)

Dibasic sodium phosphate. Adjust pH as needed.

Non-standard Supplies and Reagents:

Most supplies needed are already part of a well stocked lab or are easily found from any supplier, others are listed below.

For a more detailed list of reagents and equipment used for this method please see the methods section of Bird et al. eLife (2018).

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