Fe(II)-EDTA is capable of catalyzing the breakage of RNA or DNA and is a complementary method to RNase protection analysis. It has the advantage that there is little or no base sequence specificity, that its shear along the entire length of the RNA or DNA strand is almost uniform, and that it can be used to explain the more advanced folding of some RNAs. This experiment is derived from "RNA Lab Guidebook" edited by Xiaofei Zheng.
Operation method
Scheme 5.9 Fe(II)-EDTA protection assay
Principle
Fe(II)-EDTA is able to catalyze the breakage of RNA or DNA and is a complementary method to RNase protection assays, with the advantage that there is little or no base sequence specificity, that its shear along the entire length of the RNA or DNA strand is almost uniform, and that it can be used to explain the more advanced folding of some RNAs.
Materials and Instruments
RNA elution buffer Annealing buffer Thiourea Sodium ascorbate Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Water bath Vertical electrophoresis unit Dark cassette
1. Water bath.
2. Vertical electrophoresis unit.
3. dark film cassettes.
4. RNA elution buffer: 80% formamide, 40 mmol/L PIPES, 1 mmol/L EDTA, 0.4 mol/L NaCL.
5. Annealing buffer: 50 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 10 mmol/L MgCl2.
6. ( NH4)2Fe ( SO4)2 solution: 10 mmol/L ( NH4)2Fe ( SO4)2 (Sigma), 20 mmol/L EDTA, shake well before use.
7. 100 mmol/L thiourea.
8. 0.9% H2O2.
9. 10 mmoI/L sodium ascorbate.
II. Methods of operation
1. Preparation of RNA: Purify the end-labeled RNA by PAGE, extract the RNA-containing gel block with RNA elution buffer, and then precipitate the RNA by ethanol.
2. Annealing: to allow RNA to form a proper structure. Adjust the concentration of RNA to 25 mmol/L with 50 μl of annealing buffer, heat at 95℃ for 2 min, and then place on ice for 1 h. The concentration of RNA should be adjusted to 25 mmol/L with 50 μl of annealing buffer.
3. Reaction: Mix 7 μl of RNA, 1 μl of 10 mmol/L ( NH4)2Fe ( SO4)2 solution, 1 μl of 10 mmol/L sodium ascorbate, and 0.9% H2O2, and incubate at 20°C for 10 min. Add 1 μl of 100 mmol/L thiourea to terminate the radical reaction.
4. Assay: The cleavage products were analyzed by 10%~15% PAGE containing 8 mol/L urea and radioautography.