RNaid^® PLUS Kit with SPIN Protocol

A. Isolation of Total RNA from Cells, Bacteria, Yeast, and Tissue

B. Isolation of RNA from Solution, Agarose, or Polyacrylamide Gels

C. Isolation of RNA from Agarose/Formaldehyde Gels

D. Purification of RNA from Transcription Reactions

Remove reagents from RNaid Plus Kit with SPIN. If a precipitate is present in the Cell Lysis Solution, heat to 50°C before use to completely dissolve; then cool to room temperature. Additional materials required: 100% ethanol, Sorvall SS-34 rotor (or equivalent) and centrifuge, microcentrifuge.

Note: Carry out all steps at room temperature unless otherwise stated.

1. Centrifuge cell suspension (see Box for "Anticipated Yields") for 5 minutes at 6,500 x g and discard supernatant.



2. Add 1 ml Cell Lysis Solution/10⁷ cells, mix with pipet tip or vortex. Transfer to new tube if applicable (i.e., if lysis volume is 500 µl or less, transfer to 1.5 ml microcentrifuge tube). Continue with step 3.

1. Centrifuge bacterial culture for 5 minutes at 6,500 x g and discard supernatant (see Box for "Anticipated Yields")



2. Resuspend pellet with up to 10⁹ cells in 100µl sterile TE buffer (10 mM TrisHCl; 1 mM EDTA, pH 7.4) and add a small crystal of lysozyme. Incubate on ice for 5 minutes; then warm to room temperature and add 1 ml Cell Lysis Solution. Mix with pipet tip or vortex. Continue with step 3.

Note: Yeast cells must be lysed as spheroplasts. A Yeast Cell Lysis Kit providing all reagents and instructions is available from BIO 101 (Catalog No. 2015-400). Individual components are also available (Spheroplasting Enzyme and Solution: Catalog No. 2015-407 and 2015-406; Sorbitol: Catalog No. 2210-206; Spheroplast Test Mix: Catalog No. 2010-208).

1. Grow yeast culture in YPD medium (Catalog No. 4001-021) to a cell density of 2 x 10⁷ cells/ml (O.D.600 = 0.5-1.0; varies with strain).




Note: Do not proceed with cells at densities greater than 2 x 10⁷, as they will be difficult to spheroplast. If cell density is greater than 2 x 10⁷, dilute to < 6 x 10⁶ cells/ml and incubate until desired density is reached.




2. Centrifuge up to 5 ml of culture (1 x 10⁸ cells) at 600 x g for 5 minutes and discard supernatant. Resuspend cells in 1 ml of 0.9 M sorbitol, 0.1 M EDTA. Transfer to microcentrifuge tube and spin for 10 seconds. Discard supernatant and resuspend cells in 500µl of 0.9 M sorbitol, 0.1 M EDTA, 10 mM DTT. Add 10 µl of 20 mM CaCl₂ and 80 µl of lyticase (reconstitute lyophilized lyticase in 50 mM Tris-HCl, pH 7.5; 5 mM beta-mercaptoethanol; 20% glycerol, at a concentration of 25,000 units/ml. Freeze 80µl aliquots in liquid nitrogen and store at -70°C until use; thaw only once before using). Incubate at 37°C for 12-30 minutes and start monitoring spheroplast formation after 12 minutes. Place 20 µl of 1% SDS at one end of a glass slide and 20 µl of 1 M sorbitol at the other end. Mix 2 µl of yeast cells with each solution; cover with cover slip, and observe microscopically. Spheroplasting is sufficient when less than 5% of the cells remain intact in the SDS solution as compared to the control cells in sorbitol. Continue to incubate until 90-95% spheroplasting is reached. Continue with step 3.

Note: Fresh tissue is preferable for RNA isolation. Alternatively, tissue should be frozen immediately in liquid nitrogen and stored at -70°C. Refer to Box for "Anticipated Yields".

1. a. For fresh tissue: Add 1 ml Cell Lysis Solution/200 mg tissue and homogenize with a few strokes in a glass/teflon homogenizer.
   
   b. For frozen tissue: Cut into small pieces while frozen and immediately homogenize in Cell Lysis Solution (1 ml/200 mg tissue) in a glass/teflon homogenizer or a blender.
   
   c. For plant tissue: Pulverize in liquid nitrogen using a mortar and pestle. Then add Cell Lysis Solution (1 ml/200 mg tissue) and homogenize in a glass/teflon homogenizer or a blender.



2. Transfer to sterile tube and continue with step 3, below.
   
   

   Anticipated Yields
   Sample	Amount	Total RNA Yield
   muscle tissue	100 mg	100 to 150 µg
   liver tissue	100 mg	up to 800 µg
   fibroblasts	107 cells	50 to 80 µg
   lymphocytes	107 cells	70 to 100 µg
   epithelial cells	107 cells	100 to 120 µg
   young plant leaf	250 mg	50 to 200 µg
   mature plant leaf	250 mg	25 to 100 µg
   bacterial cells	109 cells	50 to 100 µg
   yeast	108 cells	50 to 100 µg

   
   
   
   
3. Add 1 ml of Acid Phenol per ml of cell lysate (up to 5 ml), mix or vortex.



4. Add 0.5 ml Chloroform/Isoamyl Alcohol for each 1 ml of Acid Phenol used in step 3 (up to 2.5 ml); mix or vortex.



5. Incubate on ice for 15 minutes.



6. Spin at 4°C for 20 minutes at 10,000 x g. RNA will be in the top aqueous phase, DNA and protein in the inter- and bottom phases. See Troubleshooting Guide if phases do not separate.



7. Transfer top phase to new vial. Be careful not to remove any of the interphase.



8. Extract with up to 1 ml Chloroform/Isoamyl Alcohol (see initial preparation instructions) and spin for 2 minutes. Remove top phase to new vial. Be careful not to remove any of the interphase.



9. Binding of RNA to the RNaid Matrix will be affected by the ionic strength of the solution. To minimize binding of tRNA and thus enrich for mRNA, do not add RNA Binding Salt and proceed to step 10 immediately. If total RNA including tRNA is desired, add an equal volume of RNA Binding Salt. A white precipitate will form when RNA Binding Salt is added. It will be washed away in step 13 below.



10. Add RNaid Matrix: Vortex RNaid Matrix vial before each use to achieve homogenous suspension of the matrix material. Use 1µl of RNaid Matrix per µg of RNA expected, but use a minimum of 5 µl. In general, 100 µl per 1 gram of tissue is sufficient. Mix or vortex for 30 seconds and incubate at room temperature for 5 minutes with occasional mixing to allow adsorption of RNA to the RNaid Matrix.



11. Spin for 1 minute in microcentrifuge at maximum speed, or at 10,000 x g to pellet the RNA/RNaid Matrix complex.



12. Transfer supernatant to new tube and save for possible re-adsorption. Briefly respin vial and remove traces of liquid with a small bore pipet tip. Resuspend white pellet in 300 µl RNA Binding Salt only if RNA Binding Salt was added in step 9 above. If RNA Binding Salt was not used in step 9, continue directly with washing step 13. Carefully resuspend pellet by stirring with pipet tip. If the sample is in a larger vial, it should at this point be transferred to a microcentrifuge vial. Spin in microcentrifuge for 1 minute and remove supernatant.



13. Resuspend white pellet in 500 µl RNAWash Solution (remember to add ethanol before first use) and mix with pipet tip. Spin in microcentrifuge for 1 minute and remove supernatant.



14. Wash pellet a second time as described in step 13 above. Briefly re-spin vial and remove traces of liquid with a small bore pipet tip.



15. Resuspend pellet in 30-100 µl (depending on size) of DEPC-Treated Water and incubate at 55°C for 5 minutes to elute RNA from RNA Matrix. Transfer suspension to a SPIN Filter and spin for 1 minute in microcentrifuge. The supernatant containing RNA will be in the catch tube.

Note: Reagents for agarose/urea gels are not included with the kit.

To pour an agarose gel in 0.5x TAE buffer containing 6M urea, prepare two solutions.

Solution A:
8M urea, dissolve in water by heating to 60°C. Cool to room temperature. Adjust pH to 3.8 with solid citric acid (approx. 0.8g citric acid/100 ml 8M urea; use free acid, not sodium salt).

Solution B:
Prepare a 4x agarose solution in 2x TAE buffer, pH 6.0. Melt agarose completely by boiling.

Mix Solution A with 1/4 volume melted Solution B and cast gel. The final concentration is 6M urea and 0.5x TAE at the desired agarose concentration. The gel will solidify within 30 to 60 minutes at 4°C. An agarose concentration of less than 1% may take overnight at 4°C to solidify. The gel will remain clear upon solidification. Load sample and run at 4°C. Heat denatured RNA sample before loading by incubation at 60°C for 10 minutes in the presence of 50% formamide, or at 80°C for 10 minutes without formamide.

1. Add 3 volumes of RNA Binding Salt and mix well.



2. Continue with step 3, page 424.

1. Excise desired RNA band from ethidium bromide-stained gel and determine approximate volume by its weight. Place gel slice in microcentrifuge tube.



2. Add 3 volumes of RNA Binding Salt (i.e., to 0.1 g gel slice, add 0.3 ml of RNA Binding Salt). Mix and incubate at room temperature for 10 minutes to dissolve agarose. Alternatively, place tube in 45-55°C waterbath to dissolve agarose more rapidly. Continue with step 3, below.

1. Excise band from ethidium bromide stained gel and determine approximate volume by weight. Place into microcentrifuge vial. If gel concentration is 10% or higher, crush or cut into small pieces. Add 3 volumes of RNA Binding Salt. Soak for 20 minutes at 60°C. Remove liquid with small bore pipet tip, avoiding gel pieces; and transfer to new vial.



2. Add 2 µl of 10% Acetic Acid (included with kit) per every 0.5 ml of liquid to change pH to 5.0-5.5 (check with pH paper). This will increase recovery efficiency. Continue with step 3, next page.



3. Estimate the amount of RNA expected and add 1 µl of RNaid Matrix for every µg of RNA. Add a minimum of 5 µl of RNaid Matrix. Mix well and allow binding of RNA to the matrix for at least five minutes at room temperature. Mix occasionally to keep RNaid Matrix in suspension during absorption.



4. Spin for 1 minute in microcentifuge at maximum speed to pellet RNA/RNaid Matrix complex. Remove supernatant and save aside; if supernatant contains residual RNA, more RNaid Matrix can be added for complete recovery. Spin pellet again briefly and remove residual liquid with small bore pipet tip.



5. Add 500 µl of RNA Wash solution (remember to add ethanol before first use) and resuspend pellet completely by mixing with pipet tip. Spin for 1 minute in microcentrifuge at maximum speed and remove supernatant.



6. Repeat washing step 5 one or two times. After last wash, spin tube again briefly and remove residual liquid with small bore pipet tip.



7. Resuspend pellet in RNase-free water (use kit-supplied DEPC-Treated Water). Use 10-20 µl per 5 µl RNaid Matrix. Mix thoroughly with pipet tip and elute RNA from matrix by incubating at 45-55°C for 5 minutes.



8. Transfer suspension to a SPIN Filter and spin for 1 minute in microcentrifuge. The supernatant containing RNA will be in the catch tube. Optional: A second elution will yield 5-15% more RNA.

Note: Reagents for agarose/formaldehyde gels are not included with the kit.

10x Gel Buffer:

• 200 mM MOPS, pH 7.0 (adjust with NaOH)
• 10 mM EDTA
• 10 mM NaOAc.

Prepare 1.2% agarose gel containing 6.6% formaldehyde and 1x gel buffer. Do not add ethidium bromide to the gel, only to the RNA sample as described below. Run gel at 3-5V/cm in 1x gel buffer with 6.6% formaldehyde at pH 7.0.

Preparation of RNA sample for gel:

• 10 µl formamide (deionized)
• 4 µl formaldehyde (37%/12.3M)
• 2 µl 10 x gel buffer
• 3 µl RNA (up to 20 µg)
• 1 µl ethidium bromide (400 µg/ml)

Heat at 65°C for 10 minutes before loading in well of agarose gel. Formamide is dense enough to allow the sample to be loaded without adding an additional dense liquid; however, a loading dye mixture can be used if preferred.

1. Excise RNA band(s) from gel after electrophoresis. Visualize RNA with longwave UV for minimal length of time while cutting gel. Determine approximate volume of gel slice(s) by weight and place slice(s) into microcentrifuge tubes.



2. Adjust the pH of the gel slice to pH 5.0 by adding 2 µl of 10% Acetic Acid (included with kit) to 1 ml of RNA Binding Salt and add 3 volumes to the gel slice (i.e., to 0.1 g gel slice, add 0.3 ml RNA Binding Salt/Acetic Acid mixture). The lower pH will optimize the binding efficiency of RNA to the RNaid Matrix. Incubate at 37°C for approximately 10 minutes with occasional mixing to melt agarose.



3. When gel is completely melted, place vial at room temperature and add 1-2 µl of RNaid Matrix per µg of RNA. Mix well and allow RNA to adsorb to the matrix for 10 minutes at room temperature with periodic mixing.



4. Centrifuge for 1 minute in microcentrifuge at maximum speed to pellet the RNA/RNaid Matrix complex. Remove supernatant to new tube and save for possible re-absorption. Briefly spin again to collect remaining liquid in bottom of the tube. Remove all traces of liquid with a small bore pipet tip.



5. Optional (recommended): Resuspend pellet in same amount of RNA Binding Salt as in step 2. to wash pellet and help remove remaining traces of agarose and formaldehyde. Mix thoroughly with pipet tip. Spin for 1 minute and remove supernatant. Spin briefly again and remove traces of liquid with small bore pipet tip.



6. Resuspend pellet in 500 µl RNA Wash Solution (remember to add ethanol before first use) by mixing with pipet tip. Spin for 1 minute and remove supernatant.



7. Repeat washing step 6 one or two times. Re-spin and remove traces of liquid as described in step 5.



8. Resuspend pellet completely in RNase-free water (use kit-supplied DEPC-Treated Water) by mixing with pipet tip. Use 10-20µl of water per 5µl RNaid Matrix. Elute RNA by incubating at 80°C for 10 minutes. Transfer suspension to a SPIN Filter and spin for 1 minute in microcentrifuge. The supernatant containing RNA will be in the catch tube.




Optional: A second elution will yield 5-15% more RNA




9. Heat eluted RNA to 80°C for 10 minutes to further dissociate residual formaldehyde from RNA. This second heating step will reverse chemical modification of the RNA caused by formaldehyde (Boedtker, H., 1967, Biochemistry 6, 2718-2727) and will render RNA biologically active as a substrate for modifying enzymes. Let cool to room temperature to allow RNA to renature, or place on ice immediately to avoid renaturation. The RNA is now ready for use in enzymatic manipulations.

Note: Reagents for transcription, template removal, and hydrolysis are not included with the kit.

1. Combine:
   
   • 5 µl 5x transcription buffer
   • (200 mM Tris-HCl, pH 7.5 at 37°C, 30 mM MgCl₂, 50 mM NaCl, 10 mM spermidine)
   • 1 µl RNase Inhibitor (1 unit/µl)
   • 1 µl 50 mM DTT
   • 1 µl each rATP, rGTP, rUTP (10 mM each)
   • 4 µl radioactively labeled rCTP (200 µCi)
   • 11 µl distilled water
   • 1 µl DNA template (1 µg/µl)
   • 1 µl RNA Polymerase (i.e. T3, T7, or Sp6 Polymerase)
   • 25 µl total reaction volume



2. Mix and incubate at 37°C for 1 hour. When completed, remove 1 °l of the reaction and determine TCA precipitable counts to calculate percent incorporation.


Removal of DNA Template and Hydrolysis of RNA



Note: Depending on the size of transcribed RNA and the purpose of use, it may be necessary to shorten the transcripts by hydrolysis with sodium hydroxide. After hydrolysis, it is crucial to neutralize the pH before purification of the RNA with RNaid Matrix. If the pH of the sample is alkaline, the RNA will not adsorb to the matrix.




3. Add 1 µl of RNase-free DNase (10 units/µl). Incubate at 37°C for 10 minutes and place on ice. If probe is to be hydrolyzed, continue with step 4. Otherwise, continue with Purification of RNA, step 7 below.



4. Add 50 µl ETS buffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA; 0.1% SDS), 1.7µl 5M NaCl, and 1µl 1M DTT. Mix.



5. Add 10 µl 2N NaOH for hydrolysis of RNA transcripts. Incubate on ice for 30 minutes for smaller transcripts (< 1kb) or for 60 minutes for larger transcripts (> 1kb). As an example, a 1kb transcript can be shortened to 150-220 bases by 30-40 minutes incubation at 4°C.



6. Warm to room temperature; then add 20 µl (2x volume) of 1M MES buffer to neutralize pH and stop hydrolysis. Add MES buffer after warming tube to room temperature to prevent precipitation. Volume is approximately 110 µl at this point.


Purification of RNA with RNaid Matrix


7. Add 3 volumes of RNA Binding Salt to the completed reaction; mix.



8. Estimate the amount of transcripts and add 1-2 µl of RNaid Matrix per µg of RNA; add a minimum of 5 µl RNaid Matrix. Mix well and incubate at room temperature for 5 minutes with occasional mixing to allow adsorption of RNA to the RNaid Matrix.



9. Spin for 1 minute in microcentrifuge at maximum speed. Remove and discard supernatant which contains most of the unincorporated label. Follow precautions and regulations for handling and disposing of radioactive materials as specified in Radioactive Materials License.



10. Wash pellet two times with 500 µl RNA Wash Solution as described in Sections A, B, and C.



11. Remove residual traces of liquid with a pipet and elute RNA with appropriate volume of RNase-free water (use kit-supplied DEPC-Treated Water) by carefully resuspending pellet and incubating at 50°C for 5 minutes. Transfer suspension to SPIN Filter and spin for 1 minute in microcentrifuge. The supernatant containing RNA will be in the catch tube.