RNAPurificationfrom1020mgParaffinembeddedTissue

實驗概要

The E.Z.N.A.^?  SQ Tissue RNA Kit is designed for isolating total RNA from animal  tissue and cultured cells. The solution based system can be easily scale  up and down based on the starting material. There is no need for  phenol/chloroform extractions, and time-consuming steps such as CsCl  gradient ultracentrifugation are eliminated.

RNA purified using the E.Z.N.A.^? SQ Tissue RNA method can be directly used for applications such as RT-PCR*, Nouthern blotting, and other enzymatic reactions.

實驗原理

E.Z.N.A.^?  SQ Tissue RNA uses a highly efficient solution based system to provide a  convenient, fast, reliable and non-toxic method to isolate high quality  RNA from various samples. Samples are first lysed with RCL buffer.  Cellular proteins and genomic DNA are removed by precipitation with PNP  Buffer while RNA will remain in solution. RNA is further purified by  isopropanol precipitation.

主要試劑

1. Isopropanol

2. 70% ethanol

主要設備

1. Microcentrifuge capable of 13,000 x g

2. Sterile 1.5 ml centrifuge tube

3. Ice bath

4. Ground pestle

實驗步驟

1. Place 10-20 mg  minced tissue into a 1.5 ml centrifuge tube. Add 300ul xylene and mix  throughly by vortexing for 20 seconds. . Incubate at room temperature  for 5 minutes with constant shaking.

2. Centrifuge at 13,000 x g for 5 minutes. Carefully remove the xylene with pipette.

3. Wash the tissue sample two more times by repeating step 1-2.

4. Add 600ul ethanol into the tube and mix throughly by vortexing the  tube for 10 seconds. Incubate at room temperature for 5 minutes.

5. Centrifuge at 13,000 x g for 3 minutes. Discard the ethanol.

6. Wash the sample with ethanol 2 more times by repeating step 4-5 twice.

7. Add 600 ul of RCL Buffer to the tube. Homogenize throughly with  5-10 strokes using a microfuge tube pestle (Product # SSI-1015-39).

8. Add 200 μl of PNP Buffer to the cell lysate. Mix the sample gently by inverting the tube 10 times.

9. Place the tube on ice for 5 minutes.

10. Centrifuge at max speed (13,000 x g) for 3 minutes at room  temperature. The precipitated protein and DNA will form a tight pellet.

11. Transfer the supernatant to a new sterile 1.5 mL centrifuge tube  that containing 600ul of 100% isopropanol. If the RNA yield is expected  to be low, add total 1ìg Linear Polyacrylamide (Cat# PR033) or glycogen  (Cat # AC122) to the 100ul isopropanol.

12. Gently mix the solution by inverting the tube 30-40 times.

13. Centrifuge at 13, 000 x g for 5 minutes at room temperature.

14. Pour of the supernatant and drain the tube briefly on a clean  absorbent paper. Add 600ul of 70% ethanol and invert the tube few times  to wash the RNA pellet.

15. Centrifuge at 13, 000 x g for 2 minutes at room temperature.  Carefully pour off the ethanol. Pellet may be very loose at this point  so pour slowly and watch the pellet.

16. Invert the tube on a clean adsorbent paper and air dry the pellet for 10-15 minutes.

17. Add 100 ul of DEPC Water and vortex for 1 minutes to mix.

18. Incubate sample on ice for at least 30 minutes. Vortex sample vigorously for 10 seconds and pulse spin.

19. Store RNA at -70°C.