RNA Structure - Function Protocols: Alkaline Hydrolysis, RNA Sequencing and RNA Structure Analyses with Nucleases

This procedure provides a gel electrophoresis "ladder" of hydrolyzed RNA fragments. In the procedure, 3 different hydrolysis times are used. After the experiment, select the ladder that provides the best distribution of nucleic acids over the range of lengths needed for your experiments.

Reagents Required

• 0.1–3 µg end-labeled RNA (see the 5' end labeling procedure or the 3' end labeling procedure for protocols for end-labeling RNA).
• Yeast RNA (10 mg/ml; e.g., Cat #7118 or the Yeast RNA supplied with Ambion's RNA Grade Ribonucleases)
• 1X Alkaline Hydrolysis Buffer (e.g., 50 mM Sodium Carbonate [NaHCO3/Na2Co3] pH 9.2, 1 mM EDTA; or the Alkaline Hydrolysis Buffer supplied with Ambion's RNA Grade Ribonucleases)
• Acrylamide Gel Loading Buffer (e.g., 95% Formamide, 18 mM EDTA, 0.025% SDS, 0.025% Xylene Cyanol, 0.025% Bromophenol Blue; or Gel Loading Buffer II (Cat #8546G) supplied with Ambion's RNA Grade Ribonucleases)
• 6–20% acrylamide/7 M urea sequencing gel (the percentage of acrylamide will depend on the length of RNA to be analyzed; gel size will depend on the resolution required)

Protocol

Before use, the Inactivation/Precipitation Buffer should be completed by addition of 3.2 mL of 100% ethanol.

1. Mix 0.1-3 µg of end-labeled RNA and 3 µg of yeast tRNA in a volume not to exceed 5 µl.


2. Add sufficient 1X Alkaline Hydrolysis Buffer to bring the final volume to 15 µl.


3. Aliquot 5 µl of the RNA/buffer mixture into 3 tubes labeled 1–3.


4. Heat the tubes to 95°C.


5. After 2 min, remove Tube #1 to an ice bucket.


6. After 5 min, remove Tube #2 to at an ice bucket.


7. After 15 min, remove Tube #3 to at an ice bucket.


8. Add 10 µl of Acrylamide Gel Loading Buffer to each of the 3 tubes. For an untreated control, mix 1 µl of 5' end-labeled RNA with 8 µl of Acrylamide Gel Loading Buffer.


9. Fractionate 3 µl of the RNA from all 4 tubes using a 6–20% acrylamide/7 M urea sequencing gel.


10. Use autoradiography to visualize the fractionated RNA products.

This procedure uses RNase T1 to digest denatured RNA. RNase T1 cleaves single-stranded RNA 3' to guanosine residues. Partial digestion of 3' or 5' labeled RNA with this enzyme thus generates a ladder of G residues.

The 1X RNA Sequencing Buffer used in the procedure contains 7 M Urea to help denature RNA secondary structure. To perform the procedure, end-labeled RNA is diluted at least 5X into the Sequencing Buffer. The sample is heated to 50°C to denatures the RNA, and then incubated with RNase T1 at room temperature.

In the procedure, two different concentrations of RNase T1 are used. Tube #1 contains no RNase and represents a negative control. Non-full length bands in this tube represent cleavage products present within the RNA sample. These bands will also be present in the nuclease treated samples and should be disregarded in your analysis. Tubes #2 and #3 contain two different concentrations of RNase T1. Typically, at least one of the reactions will generate a ladder comprising digestion products from each of the guanosines in the RNA molecule. Further enzyme dilution may be necessary to achieve the optimal digestion ladder.

Reagents Required

• 0.1–3 µg end-labeled RNA (see the 5' end labeling procedure or the 3' end labeling procedure for protocols for end-labeling RNA).
• Yeast RNA (10 mg/ml; e.g., Cat #7118 or the Yeast RNA supplied with Ambion's RNA Grade Ribonucleases)
• 1X RNA Sequencing Buffer (e.g., 20 mM sodium citrate pH 5, 1 mM EDTA, 7 M urea; or the 1X RNA Sequencing Buffer supplied with Ambion's RNA Grade Ribonucleases)
• RNase T1, 1 U/µl (Cat #2283)
• Inactivation/Precipitation Buffer (provided with Ambion's RNA Grade Ribonucleases)
• Acrylamide Gel Loading Buffer (e.g., 95% Formamide, 18 mM EDTA, 0.025% SDS, 0.025% Xylene Cyanol, 0.025% Bromophenol Blue; or Gel Loading Buffer II (Cat #8546G) supplied with Ambion's RNA Grade Ribonucleases)
• 6–20% acrylamide/7 M urea sequencing gel (the percentage of acrylamide will depend on the length of RNA to be analyzed; gel size will depend on the resolution required

Protocol

Before use, the Inactivation/Precipitation Buffer should be completed by addition of 3.2 mL of 100% ethanol.

1. Mix 0.15-3 µg of end-labeled RNA and 3 µg of yeast RNA in no more than 10 µl.


2. Add sufficient 1X RNA Sequencing Buffer to bring the final reaction volume to 27 µl.


3. Aliquot 9 µl of the RNA/Sequencing Buffer mixture into three tubes labeled 1-3.


4. Heat each tube to 50°C for 5 min, then cool to room temperature.


5. Add 1 µl of RNase T1 (1 U/µl) to Tube #2 and mix by pipetting.


6. Transfer 1 µl from Tube #2 to Tube #3 and mix by pipetting.


7. Incubate all tubes at room temperature for 15 min.


8. Add 20 µl of Inactivation/Precipitation Buffer and incubate at –20°C for 15 min.


9. Pellet the RNA at 13,200 RPM for 15 min, aspirate, wash with 70% ethanol, and air dry.


10. Add 7 µl of Acrylamide Gel Loading Buffer, heat to 95°C for 5 min, then fractionate 3 µl using a 6–20% acrylamide/7 M urea sequencing gel.


11. Use autoradiography to assess the digestion products.

TOP

This procedure uses RNase A, T1 or V1 to provide information about the structure of an RNA molecule. The sequence selectivity of these enzymes is listed in Table 1. In the procedure, four tubes are prepared. Tube 1 contains a negative control of end-labeled RNA without nuclease treatment. Non-full length bands in this sample represent cleavage products present within the labeled RNA itself. These bands will also be present in the nuclease treated samples and should be disregarded in your analysis. Tubes 2, 3, and 4 contain decreasing amounts of ribonuclease. Bands evident in the samples with lower amounts of ribonuclease typically represent nucleotides that are most accessible to the nuclease. Further enzyme dilution may be necessary to achieve the optimal digestion ladder.

RNase	Cleavage Site	Ambion Cat #
A	3' of ss C's and U's	2274
V1	base-paired nucleotides	2275
T1	3' of ss G's	2283

Table 1. Selectivity of RNases A, V1 and T1.

Reagents Required

• 0.2–4 µg end-labeled RNA (see the 5' end labeling procedure or the 3' end labeling procedure for protocols for end-labeling RNA).
• Yeast RNA (10 mg/ml; e.g., Cat #7118 or the Yeast RNA supplied with Ambion's RNA Grade Ribonucleases)
• 10X RNA Structure Buffer (e.g., 100 mM Tris pH 7, 1 M KCl, 100 mM MgCl2; or the 10X RNA Structure Buffer provided with Ambion's RNA Grade Ribonucleases)
• RNase T1, 1 U/µl (Cat #2283), or RNase V1, 0.1 U/µl (Cat #2275), or RNase A, 1 µg/ml (Cat #2274)
• Inactivation/Precipitation Buffer (provided with Ambion's RNA Grade Ribonucleases)
• Acrylamide Gel Loading Buffer (e.g., 95% Formamide, 18 mM EDTA, 0.025% SDS, 0.025% Xylene Cyanol, 0.025% Bromophenol Blue; or Gel Loading Buffer II (Cat #8546G) supplied with Ambion's RNA Grade Ribonucleases)
• 6–20% acrylamide/7 M urea sequencing gel (the percentage of acrylamide will depend on the length of RNA to be analyzed; gel size will depend on the resolution required

Protocol

Before use, the Inactivation/Precipitation Buffer should be completed by addition of 3.2 mL of 100% ethanol.

1. Thaw the 10X RNA Structure Buffer and end-labeled RNA by incubating at room temperature.


2. Mix 4 µl of 10X RNA Structure Buffer, 0.2-4 µg end-labeled RNA, 4 µg yeast RNA, and Nuclease-Free Water to bring the final volume to 36 µl.


3. Aliquot 9 µl of the RNA/buffer/water mixture into 4 tubes numbered 1-4. To Tube #2 add 1 µl of ribonuclease. Mix thoroughly by pipetting.


4. Transfer 1 µl from Tube #2 to Tube #3. Mix thoroughly by pipetting.


5. Transfer 1 µl from Tube #3 to Tube #4. Mix thoroughly by pipetting.


6. Incubate all tubes at room temperature for 15 min.


7. Add 20 µl of Inactivation/Precipitation Buffer and vortex.


8. Incubate at -20°C for 15 min, centrifuge at 13,200 RPM for 15 min, aspirate, and wash the pellet with 70% ethanol.


9. Air dry the pellet and dissolve in 7 µl of Acrylamide Gel Loading Buffer.


10. Heat denature the RNA at 95°C for 5 min, then fractionate 3 µl using a 6-20% acrylamide/7 M Urea sequencing gel.


11. Use autoradiography to assess the digestion product.

TOP