E.Z.N.A.?TotalRNAMaxiKitProtocolforAnimalTissues

實驗概要

The E.Z.N.A.^?  Total RNA Maxi Kit uses HiBind? matrix spin-column technology to  isolate up to 5 mg total cellular RNA from a variety of sources  including 10¹⁰ bacterial cells, 1 g tissue, and up to 5 x 10⁸  cultured cells. Samples are lysed and homogenized in denaturing  conditions to ensure degradation of proteins and inactivation of  endogenous RNases. Binding conditions are then optimized to favor RNA  isolation before the resulting cell lysate are passed through HiBind^?  RNA Maxi spin-columns. Subsequent wash steps remove cellular debris and  trace salt contaminants to allow elution of pure RNA in DEPC-treated dH₂O.  No organic solvents are used and only short centrifugation steps using a  standard centrifuge equipped with a swinging-bucket rotor at room  temperature are needed. The kit can also be used for RNA clean-up prior  to any downstream application. Purified RNA is suitable for mRNA  isolation (Product R6511, mRNA Enrichment Kit), Northern analysis, RNase  protection assays, differential display, and reverse transcription.

主要試劑

1.  2-mercaptoethanol ($-mercaptoethanol, $-ME) is required and must be  added to TRK Lysis Buffer before use. Add 20 ul $-ME per 1 ml TRK Lysis  Buffer. The mixture should be stored at room temperature and is stable  for about 1 week.

2. 70% ethanol (in water) and absolute ethanol.

3. DEPC-treated deionized water.

主要設備

1. RNase-free 50 ml conical centrifuge tubes.

2. Centrifuge with swinging-bucket rotor at room temperature capable of 3,500 x g.

3. RNase-free pipet tips and disposable gloves.

4. Note that all centrifugation are performed using a centrifuge with  a swingingbucket rotor at 4,000 -6,000 x g at ROOM TEMPERATURE.

實驗步驟

Quantity of Starting Material: In general the binding capacity of the HiBind^?  RNA Maxi column (~5 mg RNA) will not be exceeded and between 0.1g and  1.0 g of tissue can be used. However, some tissues such as brain and  skeletal muscle tend to be rather difficult to lyse and homogenize and  can block the column, leading to lower RNA yields. With such tissues, we  recommend starting with 250-400 mg to ensure optimal column  performance. Subsequently, the starting quantity may be increased to  1.0g if favorable yield and quality is obtained. Overloading the column always leads to clogging and reduced yields.

Freezing Samples: Fresh tissue is best, but frozen samples may also  be processed. To store tissues for future use, flash-freeze under liquid  nitrogen and store at -70°C for 2-4 months. Frozen samples should be  processed directly in TRK Lysis Buffer without prior thawing. Once  homogenized in TRK Lysis Buffer, samples may be stored at - 70°C for  several months.

1. In almost every case, optimal yields are only obtained if the  tissue sample is first completely and thoroughly disrupted and  homogenized in TRK Lysis Buffer. We suggest using a mechanical rotor  homogenizer. Wearing gloves, place 250-400 mg tissue in a sterile  RNase-free 50 ml polypropylene centrifuge tube (not provided). Add 7.0  ml TRK Lysis Buffer containing 2-mercaptoethanol and homogenize for 2  minutes.

Note: Remember to add 2-mercaptoethanol to TRK Lysis Buffer before  use. Add 20 ul 2-mercaptoethanol per 1 ml TRK Lysis Buffer before use.

Tip: For difficult tissues, use up to 14.0 ml of TRK Lysis Buffer  with no more than 0.4 g tissue and extend homogenization period.

2. To obtain a cleared lysate centrifuge for 15 min at 5,000 x g. A  loose pellet usually forms accompanied by an upper lipid layer.

3. Without disturbing the pellet or the lipid layer, carefully pipet  only the cleared lysate to a fresh 50 ml polypropylene centrifuge tube  (not provided).

Tip: Transferring contaminants from the pellet or the lipid layer  will cause the spin column to clog resulting in significantly reduced  yields. If necessary, sacrifice 1-3 ml lysate to ensure optimal yield  and quality.

4. Add an equal volume (7.0 or 14.0 ml) of 70% ethanol to the cleared  lysate from step 2. If some volume is lost in prior steps, add the  appropriate amount of ethanol. Mix well by vortexing or vigorous shaking  for 1 minute. Precipitates may form upon addition of ethanol. This will  not interfere with the procedure so long as the mixture is thoroughly  and immediately mixed. Proceed to step 5 immediately.

5. Apply sample to an HiBind^? RNA maxi column assembled in  a 50 ml collecting tube (provided). The spin column has a maximum  capacity of approximately 25 ml. Cap the tube and centrifuge for 5 min  at 5,000 x g at room temperature. If more than 0.4 g tissue was used,  extend centrifugation time to 10-12 min. Discard flow-through liquid and  reuse collecting tube in step 6.

Tip: If sample volume exceeds 25 ml, load the spin column in  successive aliquots and centrifuge as above, discarding the flow-through  liquid each time.

Optional: This is the starting point for on-membrane DNase I digestion treatment. See page 9 for detail protocol.

6. Add 15 ml RNA Wash Buffer I, cap the 50 ml collecting tube and  centrifuge for 5 min at 5,000 x g at room temperature. Again discard  flow-through liquid and reuse 50 ml tube in step 7.

7. Add 10 ml RNA Wash Buffer II diluted with absolute ethanol to the HiBind^?  RNA maxi column, cap the 50 ml tube and centrifuge for 3 min at 5,000 x  g at room temperature. Discard flow-through liquid and reuse the 50 ml  tube in step 8.

Note: RNA Wash Buffer II is supplied as a concentrate and must be diluted with absolute ethanol as described on page 4.

8. Repeat step 7 with another 10 ml RNA Wash Buffer II. Discard  flow-through liquid and re-insert the spin column in the empty 50 ml  collecting tube. Centrifuge the empty column for 15 min at 5,000 x g to  dry the HiBind^? matrix. This step is critical for removing  traces of ethanol that may otherwise interfere with subsequent  downstream applications.

9. RNA elution. Transfer the HiBind^? RNA maxi column to a  new 50 ml centrifuge tube (not supplied) and pipet 1-2 ml DEPC-treated  water directly onto the matrix. For expected RNA yields greater than 1  mg use 1.25 ml of water. Cap the tube and allow the matrix to soak for 1  min. Centrifuge for 5 min at 5,000 x g.

Tip: Yield will be increased by 30%-70% by repeating the elution with  a second volume of DEPC-treated water. For a higher final RNA  concentration, this second elution may be performed using the first  eluate. However, this will result in a ~30% lower overall yield.

Note: For laboratory centrifuges not capable of achieving 5,000 x g  step 8 may not completely dry the column. As a result traces of ethanol  may remain on the HiBind? RNA matrix and will be eluted with RNA. In  this case it is best to further purify the RNA following elution by  ethanol precipitation. Add 1/10 volume DEPC-treated 5 M ammonium acetate  followed by 3 x vol absolute ethanol. In situations were expected RNA  yields are less than 100ug or so, it is recommended to add 5 ug yeast  tRNA or 10 ug glycogen as carrier prior to ethanol precipitation. Vortex  to mix and incubate 2 hours at -70°C or overnight at -20°C. Centrifuge  at 10,000 x g for 15 min at 4°C to pellet RNA. Aspirate supernatant and  discard without disturbing pellet. Wash once with 75% ethanol and  centrifuge as before. Discard supernatant and air-dry RNA pellet briefly  before reconstituting in DECP-treated water.