RNACollection&Purificationfromfibroustissue

實驗概要

Provides an easy and fast method for isolating total RNA from fibrous tissues which contains contractile proteins, connective tissue and collagen.

主要試劑

EZgene Tissue RNA Miniprep Kit For heart, muscle, and shin tissue(R6311F)
beta-mercaptoethanol
RQ1 RNase-Free DNase(Progema,M6101)
DEPC-treated ddH₂O 
liquid nitrogen
 

主要設備

Heat block
Mortar
Centrifuge
Dancer

實驗材料

Tissue

實驗步驟

1. Determine the proper amount of starting sample. The maximun amount of tissue can be processed is 30mg per column per 500μl Buffer LY. If there is no information regarding the starting material, use 10mg per column.

2. Homogenize the sample with a rotor-staror homogenizer or liquid nitrogen. Alternatively， sample can be homogenized by syringe and needle method.(Also with mortar in liquid nitrogen).

3. Disrupt tissue with 500μl of Buffer LY(add 20μl beta-mercaptoethanol per 1ml Buffer LY before use).

4. Add 300μl DEPC-treated ddH₂O to the homogenized lysate. Mix well by flicking the tube and add 25μl of Proteinase K solution. Mix well by pipeting and incubate at 55℃ for 10 minutes.

5. Transfer the cleared lysate to a DNA Clearance column pre-insterted in a 2m column tube. Centrifuge at 13000rpm for 2 minutes. Discard the DNA Clearence column and save the flow-through.

6. Add 0.5 volume of absolute ethanol and mix throughly by vortexing. Precipitates may form after adding ethanol and that doesn't affect the isolation procedure.

7. Transfer 700μl of the sample to a RNA column and centrifuge at 12000rpm for 1 minutes at RT. Discard the flow-through and put the column back to the collection tube. Process the remaining sample through the column. Discard the flow-through liquid and collection tube.

8. Place column in a clean 2ml collection tube, and add 500μl Buffer RB. Centrifuge as above(12000rpm for 1 minutes at RT)and discard flow-through. Reuse the collection tube for next step.

9. Add 50μl DNase I(2U, RNase-free) Mixture onto the middle of the column and incubate at RT for 15minutes. Add 200μl DNase Stop Buffer onto the column and centrifuge at 13000rpm for 1 min. Discard the flow-through. Add 300μl RNA Wash Buffer(Add ethanol before use) to the column and centrifuge at 13000rpm for 1 min. Discard the flow-through.

10. Add another 500μl RNA Wash Buffer to the column and centrifuge at 12000rpm for 30s. Discard the flow-through and collection tube, put the column, with the lid open, into a new collection tube.

11. Centrifuge the column at 12000rpm for 2 min with the lid open.

12. Transfer the column to a RNase-free 1.5ml tube and add 50~100μl DEPC-treated ddH₂O to the center of the column. Centrifugr at 12000rpm for 1 min to elute the RNA. Store the RNA solution at -80℃.(A second elution may be necessary if maximun yield is desired.)

注意事項

Buffer LY contains chaotropic salt, which may form reactive compounds when combines with bleach. Do not add bleach solution directly to the preparation waste, wear gloves and protective eyewear when handling.