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MicroRNA Extraction

1.       Lyse cells or tissues with 1 ml Buffer ML. 1ml Buffer ML is sufficient for 1x 107 cultured cells, 30-50 mg animal tissue or 50-100 mg plant tissue.

  • For culture cells grown in monolayer (fibroblasts, endothelial cells, etc.), lyse the cells directly in the culture dish as follows. Aspirate culture medium completely and add Buffer ML directly to the cells. Pipette buffer over entire surface of dish to ensure complete lysis. Transfer lysate to a clean 1.5 ml microtube and proceed to step 2 below. (This method is preferable to trypsinization followed by washing because it minimizes RNA degradation by nuclease contamination.)
  • For cells grown in suspension cultures, pellet cells at no greater than 1,500 rpm (400 x g) for 5 min. Discard supernatant, add Buffer ML, lyse by vortex or pipetting up and down, and transfer to a clean 1.5 ml microtube. Proceed to step 2.
  • For tissue sample, determine the size of the sample and homogenize by using liquid nitrogen method or syringe method. Unless using liquid nitrogen, homogenize samples directly in Buffer ML Reagent and proceed to step 2.
2.       Incubate the tube contains homogenate at room temperature for 2-3 min.
 
3.       Add 0.2 ml chloroform per 1 ml Buffer ML. Cap sample tubes securely and shake vigorously for 15 s. Incubate on ice for 10 min.
 
4.       Centrifuge at 12,000 x g for 15 min at 4oC. The mixture separates into a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA remains entirely in the aqueous phase.
 
5.       Transfer no more than 80% of the aqueous phase to a new 2.0 ml tube; add 1/3 volume 100% ethanol. Vortex to mix well.
 
 Removal of Large RNAs (>200 nt)
 
6.       Apply no more than 700 µl of the mixture from step 5 onto ezBindTM RNA Mini column. (Larger volumes can be loaded successively.) A precipitate may form on addition of ethanol in step 5. Vortex and add the entire mixture to the column. Centrifuge at 10,000 x g for 30-60 s at room temperature. Transfer the flowthrough into a new 2 ml tube for small RNA Isolation.
 
7.       Repeat step 6 by loading the remaining sample to the column. Centrifuge as above and transfer the flow-through into the same 2 ml tube used at step 6 for small RNA isolation.
 
Purification of MicroRNAs
 
8.       Measure the volume of the flow through from step 6-7. Add 2/3 volume 100% ethanol and vortex to mix well.
 
9.       Apply the mixture from step 8 into an ezBindTM MicroRNA column, centrifuge at 10,000 x g for 30-60 s. Discard the flowthrough liquid.
 
10.   Repeat step 9 by loading the remaining sample to the column and centrifuge as above. Discard the flowthrough liquid.
 
11.   Place column in the same 2 ml collection tube, and add 500 µl RWB Wash Buffer (Add ethanol before use). Centrifuge as above and discard flowthrough. Reuse the collection tube in step 12.
 
12.   Wash column with 500 µl RWB Wash Buffer. Centrifuge and discard flow-through.
 
13.   Centrifuge the spin column with the lid open for 2 min at full speed (13,000 x g) to completely dry the ezBindTM matrix.
 
14.   Transfer the column to a clean 1.5 ml microtube; add 30-50 µl DEPC-treated water directly onto column. Let the column sit at room temperature for 2 min and centrifuge for 1 min at full speed. Pre-heating the water to 70oC may increase yields.
 
 Isolation of Large RNA from ezBindTM RNA Mini Column
 
1.      Add 300 µl RNA Wash Buffer into the ezBindTM RNA Mini Column. centrifuge at 10,000 x g for 30-60 s and discard flow-through.
 
2.       Add 400 µl RNA Wash Buffer. Incubate at room temperature for 5 min. Centrifuge as above and discard flow-through.
 
3.       Add 500 µl RWB Wash Buffer. Centrifuge as above and discard flowthrough.
 
4.       Wash column with 500 µl RWB Wash Buffer. Centrifuge and discard flow-through. Then with the collection tube empty, centrifuge the spin column for 2 min at full speed (13,000 x g) to completely dry the ezBind matrix.
 
5.       Transfer the column to a clean 1.5 ml microtube; add 30-50 µl DEPC-treated water directly onto column. Let the column sit at room temperature for 2 min and centrifuge for 1 min at full speed.

 

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