NON-RADIOACTIVE RNA NORTHERN BLOT ANALYSIS

Kelly Zarka, MSU Potato Breeding and Genetics A. General Remarks Regarding Controlling Ribonuclease Activity and RNA Handling: Ribonucleases (RNases) are active enzymes that in most cases do not require cofactors to function. RNases are extremely stable and are difficult to inactivate. Even trace amounts will completely destroy RNA in a short time. It is extremely important that a RNase free environment is created before beginning the experiments. In order to create and maintain this RNase free environment the following precautions must be taken: 1. General Handling: Proper microbiological (aseptic) technique should always be used when working with RNA. Hands and dust particles contain bacteria and molds and are the most common sources of RNase contamination. Always wear latex gloves while handling reagents and RNA samples. Change gloves frequently and keep tubes closed whenever possible. During the procedure, work quickly and keep everything on ice when possible to slow any endogenous or residual RNAses. 2. Disposable Plasticware: It is recommended to use sterile, disposable polypropylene tubes during the experiment. They do not require pretreatment and are RNase free. Pipet tips should be purchased in racks NOT bags (bulk form) and designated for RNA use only. 3. Glassware/Metals/Mortar and Pestles: General glassware and metal items are contaminated with RNases and should always be treated by baking at 1800C for at least 8hrs or overnight. Cover the opening of the glassware or wrap the metal (forceps, spatulas, etc.), mortar and pestle with aluminum foil before baking. 4. Non-Disposable Plastics: Some of the plasticware needed during the experiment is not disposable such as trays, stir bars, screw caps, etc. These will melt if baked. Alternatively, they are rinsed thoroughly with a 0.1 M NaOH 1mM EDTA solution followed by thoroughly rinsing with RNAse free water.

5. Electrophoresis Tanks: Electrophoresis tanks used for RNA should be cleaned with detergent ( 0.5% SDS), rinsed with RNase free water. This should be done before running each gel. 6. Chemicals: All chemicals used for RNA experiments should be designated as for RNA only. Use only baked spatulas for these chemicals. 7. Solutions: To create RNase free water and solutions treat them with 0.1% diethyl pyrocarbonate (DEPC), or 1ml/1 Liter. DEPC is suspected to be a carcinogen and should be handled with care. It is very moisture sensitive, the stock solution should be stored at 40C in a metal container with some Drierite. When using place in hood and let it come to room temp. before opening. Add it to solutions in the hood. After adding it to the solution shake vigorously to bring the DEPC into solution. Let the solution sit at room temp. overnight and then autoclave it the next day. Autoclaving destroys the remaining DEPC that will react with nucleic acids and inhibit binding if this is not done. NOTE: DEPC will react with primary amines and cannot be used to treat Tris buffers. When preparing Tris buffers, treat the water with DEPC first, autoclave, and then dissolve the Tris to make the appropriate buffer. Solutions that cannot be autoclaved, such as non-fat milk blocking buffer, should be made with RNase free water. B. PART 1 EXTRACTION OF RNA FROM PLANT LEAF TISSUE

USING RNEASY PLANT RNA EXTRACTION KIT. Qiagen RNeasy Plant Total RNA Kit: Contains columns and solutions for 50 RNA mini-preps of up to ~100ug each. Catalog#74904 Qiagen 9600 De Soto Ave. Chatsworth CA 91311 RNeasy Total RNA Purification Protocol: 1) Preparations for extraction. a. Obtain about a liter of liquid nitrogen. This is enough for about 6 or more extractions. b. Obtain freshly harvested plant tissue. Choose very young healthy leaves. c. Weigh out 100mg of tissue from each plant. d. The kit contains two kinds of Lysis Buffers RLT and RLC that contain guanidinium isothiocyanate or guanininium hydrocloride, respectively. For potato plants use the RLT lysis buffer. Before use 2-mercaptoethanol must be added to Lysis Buffer. (10ul per 1ml of Lysis Buffer). 500ul of lysis buffer is needed for each extraction. 2) Grind tissue using a mortar and pestle. Precool a mortar and pestle by adding liquid nitrogen. Add the plant tissue to the puddle of liq. nitrogen in the mortar. Once the tissue is frozen work quickly before the tissue has a chance to thaw. Begin grinding the frozen tissue to a fine powder. Grind fast just as the liquid nitrogen disappears. IMMEDIATELY add 500ul of the Lysis Buffer containing 2-mercaptoethanol to the mortar containing the ground frozen tissue. Continue grinding the tissue with the Lysis Buffer for ~1 min. 3) Apply lysate onto QIAshredder and spin 2 min at full speed. Apply lysate from step 2 to a QIAshredder (lilac spin column) and centrifuge for 2 min at full speed. This spin assures the separation of all cell debris. Most of the cell debris is retained in the QIAshredder spin column. A small amount of cell debris will pass through the column and form a pellet in the collection tube. Be careful not to disturb this pellet in step 4. 4) Transfer flow-through fraction from QIAshredder to a new tube and add 225ul ethanol. Transfer flow-through fraction from QIAshredder to a new 1.5ml microfuge tube (not supplied with kit). Be careful not to disturb the cell debris pellet. Add 225ul ethanol to the flow-through fraction and mix by pipetting. Do not centrifuge this mixture. 5) Apply sample from step 4 to RNeasy spin column (Pink spin column).

Centrifuge ~15 sec at 8,000G (10,000rpm)[we do not have a low speed centrifuge use the one in the Beet lab]. This spin can go longer if not all of the liquid goes through. Discard the flow through and reuse the microfuge tube for step 6. 6) Wash with 700ul Wash Buffer RW1. Pipet 700ul Wash Buffer RW1 onto spin column, centrifuge and discard flow through as in step 5. 7) Wash with 500ul Wash Buffer RPE. Note: RPE buffer is supplied as a concentrate if you are using a new kit you must add 4 volumes of ethanol before use to obtain a working solution. Indicate on the bottle that it has been done. Place spin column in a NEW 2 ml microcentrifuge tube (supplied with kit). Pipet 500ul Wash Buffer RPE onto spin column, centrifuge as in step 6, discard flow-through and reuse the microfuge tube in step 8. 8) Wash with 500ul Wash Buffer RPE and centrifuge spin column 2 min. to dry the membrane. Pipet 500ul Wash buffer RPE onto spin column and centrifuge 2min at FULL speed. It is important to dry the membrane of the RNeasy spin column because residual ethanol may interfere with elution of RNA. 9) Elute the RNA with 2 X 30ul of water. Transfer the spin column to a new 1.5 ml microfuge tube (supplied with kit) and elute the RNA with 30ul of DEPC-treated water. Make sure that the DEPC-treated water is pipetted directly on the membrane of the spin column. Centrifuge 60 sec at 10,000rpm. Repeat with another 30ul. 10) Storage, Quantitation, and Determination of Quality of total RNA. Storage: Total RNA should be stored at -700C and should be stable for over 1 year. However if the RNA is not pure there is a chance that over time and freeze/thaw cycles some degradation could occur. Quantitation: The concentration and purity of RNA can be determined by measuring the absorbance at 260 nm and 280 nm in a spectophotometer. An absorbance of 1 unit at 260nm corresponds to 40ug of RNA per ml (A260 = 1 = 40ug/ml) The ratio between the absorbance values at 260 and 280nm give an estimate of RNA purity. Pure RNA has an A260/A280 ratio of 1.7-2.0. Note: When measuring RNA samples be sure that the cuvettes

are RNAse-free. This can be done by first washing with 0.1 M NaOH, 1mM EDTA and then rinsing with DEPC ddH20. B. PART 2 EXTRACTION OF RNA FROM PLANT TUBER TISSUE Combination of Qiagen RNeasy kit and an extraction protocol from Rapid and Efficient Method for RNA Isolation From Plants With High Carbohydrate Content. Focus:vol.13 page 103. 1993. Kai-Wun Yeh (author). 1) Weigh 1 gram of tuber tissue. Avoid green tubers. Use a coring tool and weigh 1g of a (freshly harvested is preferred but RNA has been successfully extracted from tissue stored 4 months at 42oF) tuber. Go to step 2 unless you are storing the tissue, than place in a labeled microfuge tube close lid and place in liquid N2. NOTE: Less RNA extracts out of frozen stored tuber tissue than fresh or stored tubers. 2) Immediately add 2ml of Tuber Extraction Buffer with FRESHLY added β-mercaptoethanol. Transfer to a 50ml conical tube. Add extraction buffer with freshly added β-mercaptoethanol at 0.1M. 3) Homogenize the sample in the 50ml conical tube with a large tip homogenizer. Our lab borrows the homogenizer from MFT lab. It has a small and a large tip but the large tip worked better with the 50ml conical. 4) Remove the homogenized sample with a 1ml blue tip pipet (cut the tip off so that the debri will transfer and transfer all the sample to 2 microfuge tubes. Centrifuge at max. speed for 2 min- 5min. This should pellet the starch and cell debri. 5) Remove supernatant and place on Qiagen lilac shredder and spin samples for 2 min on maximum speed 14,000g. Transfer the supernatant to a fresh microfuge tube and apply the remaining supernatant to the same shredder column until the entire sample has gone through the column. 6) Continue with the rest of the Qiagen RNeasy protocol. Be sure to add the correct amount of ETOH to each sample before it is added to the PINK column. Only use one PINK column for each plant line. The sample will have to be added in increments until it has all gone through the column. Tuber Extraction Buffer:

7.5 M Guanidinium-HCl 25mM Sodium Citrate 0.5% w/v Lauryl Sarcosinate Store at room temperature. 0.1M β−mercaptoethanol added freshly before use. C: Electrophoresis of RNA through Gels Containing Formaldehyde Caution: Formaldehyde vapors are toxic. Solutions containing formaldehyde should be prepared in a chemical hood and if possible run the electrophoresis in the hood as well. 1. Prepare a Formaldehyde-Agarose gel. For a 175ml 1% gel poured into a 15X15cm gel tray: Melt 1.75g of agarose in 124.45ml ddH2O in a 1L flask for ~3min in a microwave. Cool gel to 600C by placing it in a water bath set at that temperature, for at least 20min. To the melted agarose add 17.5ml of 10X MOPS buffer and 31.3ml of formaldehyde (in a chemical hood) and swirl to mix avoiding bubbles. Cast the gel in a chem. hood and allow the gel to set 45min. at room temp. 10X MOPS 1Liter 200mM Morpholinopropansulfonic acid 41.86g 50mM Sodium Acetate 4.10g 10mM EDTA 3.72g ddH2O 800ml pH 7.0 then bring to 1 liter Cover bottle in aluminum foil. DEPC treat 1ml/liter O/N Autoclave Solution turns to a light straw yellow color after autoclaving. Store RT for ~6 months. If solution turns a dark yellow do not use. For gels of different sizes: Final concentrations of buffers are as follows: 1X MOPS Running Buffer

2.2M Formaldehyde (Formaldehyde is obtained from the manufacturer in a 12.3M solution) 2. Preparing RNA samples for electrophoresis. 2X RNA Sample buffer: (make up fresh before use) 250ul Formamide 83ul Formaldehyde 50ul 10X MOPS 0.5ul Ethidium bromide 5X RNA Running Buffer: 50% glycerol 1mM EDTA (pH 8.0) 0.25% w/v bromophenol blue DEPC treat Autoclave Store at RT. Plant RNA samples: Up to 30ug of RNA is typically run in each lane. Start with 15-20ug of RNA per lane and adjust if necessary on future gels depending on signal. Remove samples from -80oC freezer and thaw on ice. Keep samples on ice. For each sample add an equal volume of 2X sample buffer. Then add 5X running buffer equal to 1/5 of the volume. Place samples in 65oC water bath for 10min. Return samples to ice until ready to load on gel. 3. Running the Formaldehyde/Agarose gel. Once gel is set, fill tank with 1XMOPS buffer. (~ 800mls are needed for the 15cm gel box). Load samples on gel using RNase free pipet tips. Run gel at 50 volts (~6-7 hours) or O/N (18-20 hours) at 20 volts. The bromophenol blue dye front should run no further than ~1 inch from bottom of gel and no less than 2/3 of the length of the gel. 4. Viewing the Formaldehyde/Agarose gel. Remove gel from electrophoresis tank and place in a plastic tray. Remove the formaldehyde from gel by incubating the gel 2X 15min in ~250ml ddH2O (DEPC treated).

Equilibrate the gel for blotting in ~ 200ml of 20XSSC (DEPC) for 10min or until ready to blot. While this is equilibrating view the gel on a U.V. transilluminator. RNAses can still destroy the RNA inside the gel so maintain an RNAse free environment. Place a piece of plastic wrap on the surface of the transilluminator and gently put gel on top of plastic. Take photo as usual with a transpararent ruler to measure the distance of migration from the loading well. There should be 2 major bands visible with 3 smaller bands showing up lightly below that. The bands should be sharp. If they are slightly smeared than some RNA degradation has occurred but can probably still be used to blot. However, if RNA is mostly smeared than major degradation has occurred and blotting is not recommended. These major bands can be used as size standard for determining the size of the mRNA detected ladder on the northern blot. The band sizes from top to bottom are as follows: 3200kb, 1800kb, 1500kb 1300kb and 1100kb. DIG labeled RNA molecular weight markers were purchased from BMB and tested but were degraded after just a few freeze thaws. After the photo, return gel to the 20XSSC buffer, cut the left corner of the gel off to mark it and proceed with the Northern Blotting procedure. Solution for Northern Blot: 20 X SSC 3.0M NaCl 175.30g 0.3M NaCitrate 88.2g ddH20 to 1 Liter pH to 7.0 DEPC treat with 1ml/1L overnight at R.T. Autoclave D. INSTALLATION OF THE NORTHERN BLOTTING STACK 1. Wrap a plastic platform, the size or larger than the gel, with Whatman paper and place in a plastic container. Fill container 1/2 full with 20x SSC. Allow the Whatman paper to become saturated with the solution. 2. Saturate 3 pieces of Whatman paper a little larger than the size of gel with 20x

SSC. Place on top of the platform. Remove air bubbles by rolling a clean test tube over the top.

3. Place gel upside down onto the Whatman paper and remove any trapped air

bubbles under the gel by pressing with fingers of your gloved hand. (Gel is placed upside down because the front gel wells are ridged. A flat transfer surface is on the back of the gel.)

4. Cover the gel with a large piece of plastic wrap and with a razor blade cut the plastic wrap around the edge of the gel. Remove the plastic that is on the gel

leaving a frame around sides. This ensures that the capillary movement of buffer will only go through the gel.

5. Cut a piece of nylon membrane (Amersham Hybond N) the size of the gel. DO

NOT TOUCH THE MEMBRANE WITH YOUR FINGERS! High background can be caused by this. Use blunt ended forceps and only touch corners!! Wet the nylon membrane with 20x SSC. Carefully lay it on top of the gel, if an air bubble is present lift up on that part of the membrane and lay it down again. Do not touch the membrane or roll a test tube directly on it to remove air bubbles.

6. Saturate 2 pieces of Whatman paper with 20x SSC and place on top of the nylon

membrane. Make sure there are no air bubbles by rolling a clean test tube over the top.

7. Cut a stack of paper towels ∼2 inches thick to fit into the plastic tray and place dry

on top of the Whatman paper. 8. Place a flat piece of plastic on top of the blotting stack. Place on top of that a 500g-

1kg weight (e.g. a flask or bottle with water). 9. Allow transfer to proceed overnight.

E. TAKING DOWN BLOT

1. With gloved hands remove the layers of the blotting stack down to the nylon membrane. With a pencil, mark the positions of the gel wells on the nylon membrane.

2. Mark the upper left corner of gel by cutting the corner of the nylon membrane off . (Since the gel was turned over for blotting, cutting the right corner of the

membrane while it is facing the gel will mark the upper left corner of the gel.) 3. Place the membrane RNA side up onto Whatman paper. 4. To bind RNA to the membrane, EITHER place the blot between two pieces of

Whatman paper and bake at 80° C in an oven for 2 hours or use the Stratalinker (located in rm 294). Crosslink by using 200 Joules. RNA is more sensitive than DNA so it is not recommended that the autocrosslink mode be used.

5. Rinse the blot briefly with 5X SSC. Continue with prehybridization unless the blot

will be stored then allow the blot to dry on Whatman paper. 6. Store the dry blot between sheets of Whatman paper and in a plastic bag and store

at 4°C until ready for the hybridization experiment.

F. PREHYBRIDIZATION 1. Place the RNA northern blot into a small hybridization tube by carefully rolling it with the RNA side toward the inside of the tube. Use gloved hands and baked blunt ended forceps. 2. Warm 20mls of prehybridization solution to 520C. 3. Add the prehybridization solution to the blot in the tube and place tube in hybridization oven that has been warmed to 520C. MAKE SURE YOU BALANCE THE TUBE WITH ANOTHER TUBE OF EQUAL WEIGHT! 5. Prehybridize blot for at least 2hrs. Longer times are possible. G. HYBRIDIZATION 1. Prepare hybridization solution for hybridization as follows. Hybridize a 15x15cm blot with at least 6mls of solution and no more than 20mls. The amount varies depending on the amount of stock probe that you have. (20mls is best) Warm the Hybridization solution to 520C. 3. The RNA probe is not boiled to denature, as in southern DNA probes, because it is singled stranded. The final concentration of the probe should be 25ng/ml. Add this to the pre-warmed hybridization solution. Usually 1 μl of probe in 20 ml of solution. (Note: The quantitation of probe concentrations gives approximate quantities, therefore you may have to decrease or increase the probe concentration in the hybridization solution depending on the signal and background.) 4. Remove the prehybridization solution from the hybridization tube. The prehybridization solution can be reused 2 times. Store at -200C. 6. Do not let blot dry in tube. Immediately add the hybridization solution and place in hybridization oven. Make sure the tube is balanced! Incubate overnight at 520C H. STRINGENCY WASHES Set a water bath for 650C. 1. Remove tubes from oven and increase the oven temperature to 650C for future use. Remove hybridization solution. (The hybridization solution can be saved. Store in a 50ml tube labeled very clearly with the name of probe and # of times used. Reuse 1 or 2 times. To reuse thaw at R.T. then warm to hybridization temperature.)

2. Immediately wash membrane twice in the hybridization tube with about 50mls of 2X Wash solution for 15min per wash at room temp. 3. Warm 0.5X Wash solution in 650C water bath and add 50ml to membrane in hybridization tube. Place in hyb. oven which has been warmed to 650C and incubate for 15min. Remove solution and add fresh warmed solution and incubate another 15 min in the oven. 4. Proceed directly to the detection procedure. NOTE: HYBRIDIZATION AND STRINGENCY CONDITIONS: DIG-Labeled probes demonstrate the same hybridization kinetics as radiolabeled probes. Labeled probes can hybridize non-specifically to sequences that bear homology but are not homologous to the probe sequence. These hybrids are less stable than perfectly matched hybrids. They can be dissociated by performing washes of various stringencies. The stringency of washes can be manipulated by varying the salt condition and temperature. It is recommended that one should hybridize stringently, i.e. optimize hybridization conditions (increase temp., but not higher than 600C) rather than washing stringently. I. SOLUTIONS 1. Prehybridization and Hybridization solution: Note: These solutions contain SDS which denatures RNases so the blots are safe. Concentration Amount of Stock solution 5X SSC 75ml of 20XSSC 2% Block solution 60ml of 10% Block Stock Solution 0.1% N-lauroylsarcosine 3ml of 10% N-lauroylsarcosine 0.2% SDS 6ml of 10% SDS ddH2O 6ml of ddH2O (DEPC treated and autoclaved) Formamide(deionized)* 150ml of Pure Formamide 300ml Total Aliquot in 50ml tubes and store at -200C until use. RNA Probes are added just before use. *Deionizing Formamide: If using a new unopened bottle than use directly otherwise place the desired amount of formamide into a beaker in the fume hood. Add mix resin beads 15g/150ml of formamide to the beaker. Mix with stir bar for 1 hour. Filter the formamide through a piece of Whatman paper. Now it is ready to add to the Pre/Hybridization solution. 2. 2X Wash Solution: Concentration Amount of Stock solution

2X SSC 100ml of 20X SSC (DEPC treated) 0.1% SDS 10ml of 10% SDS ddH20 890ml of ddH20 (DEPC treated) 1 Liter 3. 0.5X Wash Solution: Concentration Amount of Stock solution 0.5X SSC 25ml of 20X SSC (DEPC treated) 0.1% SDS 10ml of 10% SDS ddH20 965ml of ddH20 (DEPC treated) J. DETECTION 1. After hybridization and stringency washes, rinse membrane briefly with WASHING BUFFER about 2-5 min. 2. Incubate membrane in 80mls of BUFFER 2 for 30 min. 3. Dilute anti-DIG-AP conjugate 75 mU/ml (1:10,000 dilution) in 20ml of fresh BUFFER 2. 4. Incubate membrane for 30 min in the antibody solution on a shaker either in a plastic tray or in a zip lock bag. Ensure that the solution is covering the entire blot with gentle agitation. 5. In a plastic tray wash the membrane 2X 15 min. with 100ml of WASHING BUFFER. Gentle agitation. 6. Equilibrate membrane 2-5 min. in 20 ml BUFFER 3. 7. Dilute CSPD (25mM) or CDP-STAR 1:100 in BUFFER 3. A 15X15cm blot will need 2ml of this solution. Do not reuse on RNA blots. 8. Place blot on a piece of Whatman paper to remove excess liquid. DO NOT LET DRY. Using forceps lift and place in a clear plastic sealable folder RNA side up. Pipet the diluted CSPD or CDP-STAR solution onto the blot. Carefully close the folder. Ensure that the entire blot is covered with the solution. Once closed, immediately seal the folder closed with the sealer. Incubate blot in dark for 5 min. 9. Cut open the folder. With forceps remove blot and place on a piece of Whatman paper to remove excess moisture. DO NOT LET DRY. Keep blot RNA side up.

10. Place the membrane in a new plastic folder and seal closed with the sealer. FOR CSPD incubate for 10 min at 370C (This enhances the luminescent reaction.) FOR CDP-STAR this step is not necessary. 11. Expose the blot to film. (CSPD is a chemiluminescent substrate for alkaline phosphotase that enables extremely sensitive and fast detection of biomolecules by producing visible light which can be recorded on film. A delay in reaching maximum light emission is observed however the signal persists for several days on the blot.) For RNA I usually incubated the blots for 1 hour at RT before the initial exposure to film. An exposure of 10min to 1hour should be sufficient however you might have to adjust according to the intensity of signal on your blot. CSPD reaches a peak of emission after about 12 hours. Store blot at room temp. in the dark between exposures. Once exposures are completed store blot at room temp. labeled. For CDP-STAR no incubation is required. Place the blot on film and expose for 30 min. Adjust as necessary. Blots can be stripped and reprobed. K. RIBOSOMAL RNA CONTROL When comparing the amount of RNA among different lines it is necessary to prove that an equal amount of total RNA was loaded on a gel. The ethidium bromide gives an approximate estimation of this but a more accurate comparison is to probe your blot with a ribosomal probe. There should be equal amounts of this rRNA in each sample, loading is the only variable. PROBE: pW18S.2 plasmid, prepare as a usual RNA probe, cut with BamHI and use T3 RNA polymerase. Preparing your blot: 1) Probe your blot first with the transgenic gene specific probe first. 2) After detection and a sufficient exposure has been reached remove the blot from the

plastic envelope and wash with DEPC treated H2O. 3) Place the blot in prehybridization solution and incubate at 42oC. 4) Hybridize the blot with the RNA ribosomal probe at 42oC. L. STRIPPING AND REPROBING Note: The complete stripping of a northern blot which has been hybridized with a RNA probe can be difficult because of the length of the probe and the fact that the DIG-UTP nucleotide binds tightly with RNA. If no signal was obtained and a new or different probe would like to be used then follow the procedure below. However if a strong signal or strong background was obtained then stripping could be difficult. Alkaline stripping of membranes:

1. Thoroughly rinse the membrane in water. 2. Incubate twice for 20 minutes in 0.2M NaOH, 0.1% (w/v) SDS at 370C. 3. Rinse the membrane in 2X SSC. The membrane is now ready for prehybridizarion or can be allowed to dry and then stored at R.T. for short term storage or at 40C for long term. Non-Alkaline Stripping of Blots: 1. When using DIG-labeled molecular weight standards on your blot, methods for stripping with alkaline treatments will remove the label from the standards, therefore use only the following protocol. 2. Remove the blot from the sealed folder. DO NOT LET BLOT DRY. Wash blot 1 min in ddH2O. Boil 100mls of STRIPPING solution in a beaker and once boiling pour into a plastic container. Immediately place the blot, using forceps, into boiling solution. Agitate. Leave in solution for 10-15 min. 3. Remove the blot and place in a tray with 2X SSC, enough to cover the blot. Incubate on shaker gently for 15min. 4. Place blot in a hybridization tube, immediately add the Prehybridization solution. Do not allow the membrane to dry during any stage of the stripping procedure. DETECTION SOLUTIONS: 1) MALEIC ACID BUFFER:

Concentration Amount of Stock solution 0.1M Maleic acid 11.60g 0.15M NaCl 8.77g______________ pH to 7.5 using NaOH pellets add slowly until pH is reached then add the rest of ddH2O to 1 liter. DEPC treat with 1ml/1L

Autoclave 2)WASHING BUFFER Concentration Amount of Stock solution Maleic Acid Buffer Make the same as above 0.3%Tween 20 3ml for 1 Liter 3) BLOCKING STOCK SOLUTION 10X conc. Blocking reagent from BMB, 10% (w/v) in maleic acid buffer (DEPC treated). Dissolve blocking reagent by constantly stirring on a heating block at (650C). Do not boil the solution. It is difficult to get into solution and may take several hours. Be sure that all of it has dissolved and then autoclave. Store at 40C. The solution is opaque. A much cheaper alternative to BMB blocking reagent is non-fat dry milk. To prepare dissolve 1g/100ml maleic acid buffer which was DEPC treated and autoclaved. DO NOT autoclave the milk solution. Short term storage at 40C (2-3days). 4) BUFFER 2 (make fresh for each use) Concentration Amount of Stock solution Nonfat Dry Milk 1g Maleic Acid Buffer 100ml 5) BUFFER 3 Concentration Amount of Stock solution 0.1M Tris-HCl 12.1g 0.1M NaCl 5.84g ddH2O (DEPC treated and autoclaved) 600ml pH to 9.5 add ddH2O to 1 Liter

6)STRIPPING BUFFER Concentration Amount of Stock solution 0.1%SDS 10ml of 10% SDS stock ddH2O 990ml_______________ 1 Liter 7) Anti-digoxigenin (DIG)-AP(alkaline phosphotase conjugate) Fab fragments: Roche Cat.No. 1093 274, 150 Units 8)CSPD Chemiluninescent Substrate: Roche Cat.No. 1655 884, 1ml (25mM)

NON-RADIOACTIVE DIG-RNA PROBE LABELING Kelly Zarka, Potato Breeding and Genetics

PART 1. PRINCIPLE OF DIG RNA LABELING Labeled RNA probes are synthesized by in vitro transcription of DNA, cloned downstream of T3,T7 or SP6 promoters, with T3,T7 or SP6 RNA polymerases using digoxigenin-labeled uridine-triphosphate as a substrate. DIG-labeled RNA, like DNA probes, are detected after hybridization by enzyme linked immunoassay using an antibody-conjugate and CSPD chemiluminescent detection. The RNA probes produced are highly sensitive on northern blots because they:

a. have a defined unit of length b. have single strand target specificity c. do not re-anneal like double-stranded DNA probes d. template can be easily removed by DNAse treatment PART 2. PREPARATIONS FOR DIG RNA LABELING 1. Determine which RNA strand is transcribed in the plant. RNA is transcribed from the 5’ end to the 3’ end starting at the promoter upstream from the gene of interest. Shown below is an example of the PVY gene cloned into the PBI121 vector: Nos NPT II Nos CMV PVY GENE Nos pBI121 Pro Term Pro Term vector 5’ mRNA for PVY 3’ The mRNA is transcribed from the 5’ end starting at the gene’s promoter and ending at the 3’ end of the terminator sequence. This is the mRNA transcibed in the plant which is the target RNA. In northern analysis the probe RNA should be complementary to the target RNA. In other words, the RNA probe should be transcribed from the DNA strand opposite to that normally transcribed in the plant. 3’ mRNA for PROBE 5’ 2. The DNA vector for the RNA probe. The DNA from the gene you want to make into an RNA probe must be in a vector located upstream from a T3,T7 or SP6 promoter. Be sure that the complementary strand will be transribed with the designated promoter. For example, the following steps were used in designing a DNA vector for making the PVY RNA probe: The PVY gene in pBI121 did not have a promoter for the in vitro transcription therefore it had to be cloned into a pBS vector. Nos NPT II Nos CMV PVY GENE Nos pBI121

Pro Term Pro Term vector SmaI SacI 5’ mRNA for PVY in plant 3’ SmaI /SacI fragment inserted into a pBS vector at SmaI/SacI site T7 PVY GENE T3 pBS vector SmaI SacI 3’ mRNA for PROBE 5’ In this example the T3 RNA polymerase is used to transcribe RNA from the T3 promoter to obtain a mRNA transcript complementary to the PVY RNA found in the plant. 3. Linearization of the DNA vector. To prevent mRNA from being transcribed off of unwanted sequences of the plasmid vector, restriction digestion is used to linearize the vector before the in vitro transcription of the RNA. Choose a restriction site downstream of the promoter and gene being transcribed. The restriction enzyme should leave 5’ overhangs or blunt ends. Enzymes that leave 3’ overhangs are not recommended, however, if no other enzymes are possible then it can be used, but the restriction must be followed with T4 DNA polymerase treatment which has 3’-> 5’ exonuclease activity before the invitro transcription. In the PVY example below, the pBS with PVY vector is cut at a site downstream of the promoter and gene being transcribed. The plasmid would be linearized at the sma I site to stop the transcription at the end of the PVY gene. Sma I leaves blunt ends so the linearized plasmid can then be used for the in vitro transcription. PVY GENE SmaI SacI T7 T3

pBS vector Cut plasmid with Sma I to linearize PVY GENE T3 pBS vector T7 SmaI SacI 3’ mRNA for PROBE 5’ PART 3. PROCEDURE FOR DIG RNA LABELING 1. Isolate at least 1ug of plasmid containing the gene for the RNA probe. I usually use the Promega Wizard Prep (CAT #A7500) procedure for this step, it isolates ~6ug of clean plasmid. See protocol contained in kit. 2. Digest at least 1ug of the purified plasmid with an enzyme chosen according to the information given in Part 1 and 2 of this protocol. I usually digested 3ug to be sure that I would have enough. 3. After the plasmid has been linearized it must be purified from the enzyme and buffer before it can be labeled. This can be done by phenol/chloroform extraction and ethanol precipitation. After digestion: -Increase the volume to 100ul with sterile ddH2O. -To that mixture add an equal volume of phenol/chloroform 1:1 and mix by inversion. (Note the phenol should be Tris buffered) -Centrifuge 5min in centrifuge. -Transfer the top aqueous layer to a fresh tube. -Add 1/10th the volume of 3M NaAcetate pH 5.0 and mix. -Add 2X the volume of ETOH and mix. Place on ice for 10 min. -Centrifuge 30min.

-Remove all of the ETOH solution, avoiding the DNA pellet. -Add 100ul of ice cold 70% ETOH and centrifuge for 30 sec to wash the pellet. -Let pellet dry for 15 min. Resuspend pellet in 17ul of DEPC treated ddH2O. -Take a spectophotometer reading at O.D. 260 using 2ul to determine concentration of the purified digested fragment. For the RNA labeling you will need 1ug of DNA. Slightly smaller or larger amounts can still be used. 4. RNA labeling. Add the following to a microfuge tube on ice: 1ug of purified linearized DNA (bring up to 15ul with DEPC treated ddH2O) To the 15ul add: 2ul 10X transcription buffer (vial 8) 2ul NTP labeling mixture (vial 7) 2ul T3,T7 or SP6 RNA polymerase (choose the correct polymerase to add by following the information given in Part 1 and Part 2 of this protocol. 1ul RNase inhibitor Centrifuge briefly and incubate 2hrs at 37oC. Longer incubation does not increase the yield of labeled RNA. 5. Removing the DNA template. After the 2hr incubation above add 2ul of DNase I, RNase free (vial 9) and incubate for 15 min at 37oC. Stop the reaction by adding 2ul of DEPC treated 0.2M EDTA. STORE DIG RNA LABEL IN -80oC UNTIL READY FOR USE. PART 4. ESTIMATING THE YIELD OF DIG-LABELED RNA PROBES 1. Make serial dilutions of the DIG-Labeled control in DEPC treated ddH2O according to the following dilution scheme: Labeled Control Stepwise Final Total RNA Dilution Conc.(tube) Dilution 0.1ug/ul 2ul in 18ul ddH2O 10ng/ul(A) 1:10 10ng/ul 2ul in 18ul ddH2O 1ng/ul(B) 1:100 1ng/ul 2ul in 18ul ddH2O 100pg/ul(C) 1:1000

100pg/ul 2ul in 18ul ddH2O 10pg/ul(D) 1:10000 10pg/ul 2ul in 18ul ddH2O 1pg/ul(E) 1:100000 A->E are the Control dilutions which will be used as standards to quantify your labeling reaction. These samples can be stored at -800C for continual use. 2. Dilute your probe labeling reaction by making a serial ten-fold dilution. Dilution series for probe labeling reaction: Amount of DEPC treated Tube ddH2O Total Dilution Probe 2ul in 18ul ddH2O 1:10(A) (A) 2ul in 18ul ddH2O 1:100(B) (B) 2ul in 18ul ddH2O 1:1000(C) (C) 2ul in 18ul ddH2O 1:10000(D) (D) 2ul in 18ul ddH2O 1:100000(E) 3. Spot 1ul of each of the dilutions made in step 1 and 2 onto a small piece of nylon membrane, marking the membrane with a pencil to identify each dilution. Mix the dilutions very well just before spotting on the membrane. 4. Fix the nucleic acids to the membrane by crosslinking with the Stratalinker set at the autocrosslink mode. 5. Wet the membrane with 50ml of Washing Buffer. 6. Incubate the membrane in Buffer #2 for 5min at room temperature. 7. Dilute anti-DIG-alkaline phosphatase antibody 1:5000 in Buffer #2 10ml. Place membrane in a small zip lock bag with the 10mls of antibody solution. Incubate the membrane for 10 min. on shaker with gentle agitation. The diluted antibody must cover the entire blot. 8. Remove membrane from bag and place in a plastic tray. Wash the membrane 2X 5min in Washing Buffer at room temp. While this is incubating mix 45ul of NBT solution and 35ul X-phosphate solution in 10ml of Detection Buffer. This freshly prepared color substrate solution should be protected from light until use.

9. Incubate the membrane in Detection Buffer #3 for 2 min. Now place the membrane in a zip lock bag and add the diluted color substrate solution to the bag, seal and store in the dark. (Place in a drawer at room temp.) Ensure that the solution is covering the membrane and do not shake it. Let the color development occur in the dark for 30-60min. 10. When the desired spots appear in sufficient intensities, stop the reaction by washing the membrane with Buffer #4 for 5min. Let air dry and do not expose to the light. 11. Compare the spot intensities of the control and experimental dilutions to estimate the concentration of the experimental probe. For example, if the spot intensity of the control spot C (10pg labeled control DNA) is equal to the intensity of the D spot of your unknown probe which is diluted 1:1000(=103) than calculate the amount of DIG-labeled probe DNA to be as follows: 100pg/ul X 103 = 100,000pg/ul The total yield of the RNA labeled is the concentration of the DIG labeled probe multiplied by the volume of the probe suspension which is 24ul after addition of DNase and EDTA. If labeling reaction volume was in 24ul then: 100,000 pg/ul x 24ul = 2,400,000pg or 2.4ug of labeled RNA Use this as a guide to figure out your concentration. The amount of newly synthesized labeled RNA depends on the amount, size and purity of the template DNA. 12. Proceed to the Hybridization protocol. Solutions for Estimating Yield of Labeled Probe. DIG Nucleic Acid Detection Kit, Boehringer Mannheim Cat. No. 1175 041 includes: Labeled control DNA ( Cat. No.1585 738) Anti-DIG-AP conjugate ( Cat. No.1093 274) NBT ( Cat. No. 1 087 479) X-Phosphate[BCIP] ( Cat. No. 1 017 365) Blocking Reagent ( Cat. No. 1096 176) Use Nonfat Dry Milk instead its much less expensive and works the same. These items are available separately see cat. no. above. Additional solutions: some are the same as the chemiluminescent detection solutions

1. MALEIC ACID BUFFER

Concentration Amount of Stock solution 0.1M Maleic acid 11.60g 0.15M NaCl 8.77g__________________ pH to 7.5 using NaOH pellets add slowly until pH is reached then add the rest of ddH2O to 1 liter.

2.WASHING BUFFER Concentration Amount of Stock solution Maleic Acid Buffer Make the same as above Tween 20 0.3% 3ml for 1 Liter

3.BLOCKING STOCK SOLUTION 10X conc. Blocking reagent from BMB, 10% (w/v) in maleic acid buffer. Dissolve blocking reagent by constantly stirring on a heating block at (650C). Do not boil the solution. It is difficult to get into solution and may take several hours. Be sure that all of it has dissolved and then autoclave. Store at 40C. The solution is opaque. A much cheaper alternative to BMB blocking reagent is non-fat dry milk. To prepare dissolve 10% (w/v) in maleic acid buffer. DO NOT autoclave this solution. Aliquot and store at -200C. Short term storage at 40C ( 2-3days).

4. BUFFER #2 (make fresh for each use) Concentration Amount of Stock solution Nonfat Dry Milk 1g Maleic Acid Buffer 100ml 5. DETECTION BUFFER #3 Concentration Amount of Stock solution 0.1M Tris-HCl 12.1g 0.1M NaCl 5.84g ddH2O 600ml

50mM MgCl2 (DEPC treated and autoclaved) (This is not added in the Buffer 3 solution for the Northern Blots it is only needed for color detection not chemiluminescent)

25ml of 2M stock MgCl2_ pH to 9.5 add ddH2O to 1 Liter 6. BUFFER #4 Concentration Amount of Stock solution 10mM Tris-HCl 10ml of 1M pH 8.0 1mM EDTA 2ml of 0.5M pH 8.0 ddH2O 988ml of ddH2O_____ 1 Liter