RNaid^® Kit Protocol
Including "with SPIN" Option
Revision No. 1109-999-6D01W

The RNaid Kit with SPIN contains all of the solutions and reagents, except for ethanol, necessary for the isolation and purification of RNA in twenty minutes from agarose and polyacrylamide gels, from solutions, and from unincorporated radioactive label (see Protocols). The resulting RNA is suitable as a substrate for multiple enzymatic manipulations including reverse transcription, RNase protection assays, and in vitro translation.

For isolation of total RNA from cells and tissues, see the RNaid Plus Kit (catalog #1009-200, pg 90) which contains all of the ingredients of the RNaid Kit plus reagents for the isolation of RNA from tissue or cells. Also, the new Total RNA SAFEKit (see pg 94) is ideally suited for isolating RNA from any tissue or cell type without the use of phenol, chloroform, or guanidine chaotropes.

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

B. Isolation of RNA from Agarose/Formaldehyde Gels

C. Purification of RNA from Transcription Reactions

D. Troubleshooting Guide

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

Solution A: Dissolve 8M urea in water by heating to 60°C. Cool to room temperature and adjust pH to 3.8 with solid citric acid (approximately 0.8 g 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, below.

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 5-55°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 while avoiding gel pieces; and transfer to new vial.



2. Add 2 µl 10% Acetic Acid (included with kit) for every 0.5 ml liquid to change pH to 5.0-5.5 (check with pH paper). This will increase recovery efficiency.



3. Estimate the amount of RNA expected and add 1 µl RNaid Matrix for every µg of RNA (add a minimum of 5 µl 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 adsorption.



4. Spin for 1 minute in microcentifuge at maximum speed to pellet RNA/RNaidMatrix 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 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.




*Add 120 ml of 100% Ethanol and mix well before use.




6. Repeat washing step 5 one or 2 times. After last wash, spin tube again briefly and remove residual liquid with small bore pipet tip.



7. Resuspend pellet in DEPC-Treated Water. Use 10-20 µl per 5 µl RNaid Matrix. Mix thoroughly with pipet tip and elute RNA by incubating at 45-55°C for 5 minutes.



8. Spin tube for 2 minutes and remove supernatant with RNA. If using the SPIN option, transfer suspension to a SPIN Filter and spin for 1 minute in microcentrifuge. The supernatant containing RNA will be in the catch tube. The SPIN Filter will eliminate silica particles in the RNA sample and maximize the ability of the RNA to as much as an enzyme substrate.

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 10x 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 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 (add a minimum of 5 µl of RNaid Matrix. 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-adsorption. 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. Pulse spin and remove traces of liquid with small bore pipet tip.



6. Resuspend pellet in 500µl RNA Wash solution (remember to add 120 ml of 100% Ethanol and mix well with pipet tip before use. 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 DEPC-Treated Water by mixing with pipet tip. Use 10-20 µl of water per 5 µl RNaid Matrix. Elute RNA by incubating at 80°C for 10 minutes. Spin tube for 2 minutes and remove supernatant with RNA. If using the SPIN option, 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.

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)
   • 8 µl distilled water
   • 1 µl DNA template (1µg/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 µl of the reaction and determine TCA precipitable counts to calculate % incorporation.



Removal of DNA Template and Hydrolysis of RNA



Note: Depending on the size of the 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 purifcation 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 37µC for 10 minutes and place on ice. If probe is to be hydrolyzed, continue with step 4. Otherwise, continue with Purification of RNA.



4. Add 50 µl ETS buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% SDS), 1.7 µl 5M NaCl, and 1 µl 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 a 30-40 minute incubation at 4µC.



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 and mix. Do not precipitate or gel purify RNA prior to adding RNA Binding Salt and RNaid Matrix. Pre-purifying the "hot" RNA can lead to very tight binding to the RNaid Matrix and will be difficult to elute.



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 matrix.



9. Spin for 1 minute in microcentrifuge at maximum speed and 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 (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.



11. Remove residual traces of liquid and elute RNA with appropriate volume of DEPC-Treated Water by carefully resuspending pellet and incubating at 50°C for 3 minutes. Spin for 1 minute and remove supernatant containing labeled RNA to new tube. If using the SPIN option, transfer suspension to a SPIN Filter and spin for 1 minute in microcentrifuge. The supernatant containing RNA will be in the catch tube.