ExtractionofRNAfromFibroustissues

實驗概要

E.Z.N.A.?  MicroElute? Total RNA Kit provides a rapid and easy method for the  isolation of up to 50 ug of total RNA from small amount of cultured  eukaryotic cells, tissues such as laser dissected samples (LDS) or fine  needle aspirates (FNA). Normally, up to 5 x 10⁵ eukaryotic  cells or 5 mg tissue (amounts depend on the tissue used) can be used in a  single experiment. The kit allows single or multiple, simultaneous  processing of samples in less than 30 min. There is no need for  phenol/chloroform extractions, and time-consuming steps such as CsCl  gradient ultracentrifugation, and precipitation with isopropanol or  LiCl, are eliminated. RNA purified using the E.Z.N.A.? MicroElute? Total  RNA method is ready for applications such as RT-PCR*, Northern  blotting, poly A RNA (mRNA) purification, nuclease protection, and in  vitro translation.

實驗原理

The E.Z.N.A.?  MicroElute? Total RNA Kits combine the reversible binding properties of  HiBind? matrix, a new silica-based material with the speed of  mini-column spin technology. A specifically formulated high salt buffer  system allows RNA molecules greater than 200 bases to bind to the  matrix. Cells or tissues are first lysed under denaturing conditions  that practically inactivate RNases. After add the ethanol, samples are  then applied to the HiBind? MicroElute ? columns to which total RNA  binds, while cellular debris and other contaminants are effectively  washed away. High quality RNA is finally eluted in DEPC-treated sterile  water.

主要試劑

Rengents supplied by user:

1. 96-100% ethanol

2. $-Mercaptoethanol

3. Proteinase K (20mg/ml)

主要設備

Equipments supplied by user:

1. RNase-free filter pipette tips

2. Disposable latex gloves

3. RNase-Free 1.5 mL tubes

4. Water bath or heat block preset at 55°C

實驗步驟

1. Excise tissue from animal or from storage.

2. Weight up to #5 mg tissue and immediately place it into a 1.5 ml microcentrifuge tube for disruption and homogenization.

3.  Add 200 ul TRK Lysis Buffer and disrupt tissue and homogenize lysate in  TRK Lysis buffer by using methods described on page 4. Remember to add  20 ul of 2-mercaptoethanol per 1 mL of TRK Lysis Buffer before use.

Note:  Incomplete homogenization will cause clogging of the spin column and  lead to significantly lower yield. Generally, disruption and  homogenization by using mortar and pestle or needle and syringe can  generate lower yield. It is recommenced to use a rotor stator  homogenizer or beads milling methods for animal tissues.

4.  Pipet 200 ul RNase-Free water to each homogenate. The add 5 ul  Proteinase k (20 mg/ml) and mix through by pipetting or vortexing.

5. Incubate at 55°C for 10 minutes.

6. Centrifuge at $13,000 x g for 3 minutes at room temperature.

7.  Transfer the supernatant (about 600 ul) to a new 1.5ml centrifuge tube  and add 0.5 volume of absolute ethanol (96-100%). Mix throughly by  pipetting or vortexing.

8.  Place HiBind? MicroElute? RNA column into a 2 ml collection tube  (supplied with kit) and apply the mixture from step 7 (including any  precipitate) to the column. Centrifuge at 10,000 x g for 30 seconds.  Discard the flow-through and collection tube.

9.  Place column in a clean 2 ml collection tube, and add 400 ul of RWC  Wash Bufffer. Centrifuge at 10, 000 x g for 30 seconds. Discard  flow-through and reuse the collection tube.

10. DNase I Digestion (Optional): This is point to start On-membrane DNase I digestion. (See detail procedure on page 14).

11.  Place column in the same 2 ml collection tube and add 500 ul RWB Wash  Buffer diluted with ethanol. Centrifuge at 10, 000 x g for 30 seconds.  Discard the flow-through and re-use the collection tube. Reuse the  collection tube in step 12.

Note: RWB Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for instructions

12.  Wash column with a second 500 ul of RWB Wash Buffer as in step 11.  Centrifuge for 30 seconds at 10,000 x g and discard flow-through. Then  with the collection tube empty, centrifuge the column for 2 min at full  speed ($13,000 x g ) to completely dry the HiBind? matrix.

13.  Elution of RNA. Transfer the column to a clean 1.5 mL microfuge tube  (not supplied with kit) and elute the RNA with 15-20 ul of DEPC-treated  water (supplied with kit). Make sure to add water directly onto column  matrix. Let it sit at room temperature for 2 minutes. Centrifuge 1 min  at maximum speed.

RNA may be eluted with a smaller (<15ul) volume of water to get  higher RNA concentration. While reduced elution volume decrease total  RNA yield. The total yield will be 20-30% less when the elution volume  is less than 10ul.

注意事項

Equilibrate  samples and TRK Lysis Buffer to room temperature before start. All steps  must be carried out at room temperature. Work quickly, but carefully.