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E.Z.N.A.® HP Total RNA Kit Spin Protocol Eukaryotic Cells and Tissues

互联网2013-11-13

1542

实验原理

The E.Z.N.A.® HP Total RNA Kit uses the reversible binding properties of HiBind® matrix, a new silica-based material. This is combined with the speed of mini-column spin technology. A specifically formulated high salt buffer system allows more than 100 ug of RNA molecules greater than 200 bases to bind to the matrix. Cells or tissues are first lysed under denaturing conditions that practically inactivate RNases. Samples are then applied to the HiBind® spin 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.

实验试剂

Materials supplied by user

1. 2-mercaptoethanol

2. Absolute ethanol (96-100%)

3. Sterile RNase-free pipette tips and microcentrifuge tubes

实验步骤

Procedure of Cells:

1. Lyse cells (#1 x 10⁷ ) with 500 ul of Buffer GTC in a microfuge tube. For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add the appropriate volume of Buffer GTC and vortex or pipet to mix. Remember to add 20 ul of 2-mercaptoethanol per 1 ml of Buffer GTC before use.

For tissue culture cells grown in monolayer (fibroblasts, endothelial cells, etc.), lyse the cells directly in the culture vessel as follows. Aspirate culture medium completely and add Buffer GTC directly to the cells. Use 700 ul for T35 flasks or 10cm dishes, and 500 ul for smaller vessels. Pipette buffer over entire surface of vessel to ensure complete lysis. Transfer lysate to a clean 1.5 ml tube 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 (400xg) for 5 min. Discard supernatant, add Buffer GTC, lyse by vortexing or pipetting up and down, and transfer to a clean 1.5 ml tube. Proceed to step 2.

2. Homogenize the lysate according to step 1) or 2).

See ‘Disruption and Homogenization of samples’ on page 4 for more details on homogenization. If processing #1x 10⁵ cells, homogenize by vortexing for 1 min. Incomplete homogenization leads to significantly reduced RNA Yield and can cause clogging of column.

1) Homogenize the lysate for 30 seconds using a rotor-stator homogenizer . Proceed to step 3.

2) Pass the lysate at least 5 times through a blunt 20-gauge needle (0.9 mm diameter) fitted to an Rase-Free syringe. Proceed to Step 3.

3. Transfer the lysate into a gDNA Removal Column placed in 2 ml collection tube. Centrifuge at 14,000 x g for 3 minutes at room temperature. Transfer the flow-through lysate into a new 1.5 ml tube. Proceed Step 4.

Procedure of Tissues:

1. Lyse tissues (#30 mg) with 500 ul of Buffer GTC in a microfuge tube. Remember to add 20 ul of 2-mercaptoethanol per 1 ml of Buffer GTC before use. 500ul of Buffer GTC is sufficient approximately 30 mg disrupted tissue (~3 mm cube). For difficult tissues, greater than 20 mg tissue, use 700 ul of Buffer GTC. However, use no more than 40 mg tissue when the recommended maximum is exceeded.

For tissue samples, homogenize using one of the methods discussed on page 4. Unless using liquid nitrogen, homogenize samples directly in Buffer GTC/2- Mercaptoethanol and proceed to Step 2.

2. Centrifuge at maxi speed (>14,000 x g) for 5 min at room temperature.

3. Transfer the supernatant into a gDNA Removal Column placed in 2 ml collection tube. Centrifuge at 14,000 x g for 2 minutes at room temperature. Transfer the flow-through lysate into a new 1.5 ml tube.

Note: In some preparations, a fatty upper layer will form after the centrifugation, will form, transferring any pellet or fatty layer may reduce the RNA yield or clog the column.

Total RNA Isolation:

4. Add 0.5 volume (250ul or 350ul) of absolute ethanol (room temperature) to the lysate and mix well by pipetting.

5. Apply the sample onto HiBind® RNA Mini column. The maximum capacity of the spin cartridge is 750ul. (Larger volumes can be loaded successively.) A precipitate may form on addition of ethanol in step 4. Vortex and add the entire mixture to the column. With the spin column inside a 2 ml collection tube (supplied with kit), centrifuge at 10,000 x g for 30-60 seconds min at room temperature. Discard flow-through and proceed to step 6.

6. Place column in a new 2 ml collection tube and add 300ul RNA Wash Buffer I. Centrifuge as above and discard flow-through. Reuse the collection tube in step 7. If on-membrane DNase I digestion is desired, proceed to step 7, otherwise go to step 8.

7. DNase I digestion (Optional)

Since HiBind® RNA resin and spin-column technology actually removes most of DNA without the DNase treatment, it is not necessary to do DNase digestion for most downstream applications. However, certain sensitive RNA applications might require further DNA removal. Following steps provide on-membrane DNase I digestion:( see DNase I cat.# E1091for detail information)

1) For each HiBind® RNA column, prepare the DNase I digestion reaction mix as follows:

OBI DNase I Digestion Buffer 73.5 ul

RNase-free DNase I (20 Kunitz unites/ul) 1.5 ul

Total volume 75 ul

a. DNase I is very sensitive for physical denaturation, so do not vortex this DNase I mixture. Mix gently by inverting the tube. Prepare the fresh DNase I digestion mixture before RNA isolation.

b. OBI DNase I digestion buffer is supplied with OBI RNase-free DNase set.

c. Standard DNase buffers are not compatible with on-membrane DNase digestion.

2) Pipet 75 ul of the DNase I digestion reaction mix directly onto the surface of HiBind® RNA resin in each column. Make sure to pipet the DNase I digestion mixture directly onto the membrane. DNase I digestion will not be complete if some of the mix stick to the wall or the O-ring of the HiBind® RNA column.

3) Incubate at room temperature(25-30°C) for 15 minutes.

8. Place column in a new 2 ml collection tube and add 400ul RNA Wash Buffer I. (If on-membrane DNase digestion was performed in the previous step, wait at least 5 minutes before proceeding). Centrifuge as above and discard flow-through.

9. Place column in the same 2 ml collection tube and add 500 ul RNA Wash Buffer II diluted with ethanol. Centrifuge as above and discard the flowthrough. Reuse the collection tube in step 9.

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

10. Wash column with a second 500 ul of RNA Wash Buffer II as in step 9. Centrifuge as above and discard the flow-through. Then with the collection tube empty, centrifuge the spin cartridge at 10,000 x g for 2 min at room temperature to completely dry the HiBind® matrix.

11. Transfer the column to a clean 1.5 ml micro centrifuge tube (not supplied with kit) and elute the RNA with 30-50ul 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 and centrifuge at 10,000 x g for 1 min. A second elution may be necessary if the expected yield of RNA >30 ug.

Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution. Pre-heating the water to 65°C before adding to column and incubating column 5 min at room temperature before centrifugation may increase yields.

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