Dear Axel

This disease mainly occurs in sheep with high morbidity an mortality. The agent of this disease is an Orbivirus, in Reoviridae family. ds negative RNA, non enveloped virus. manual genomic purification of this virus done by this method:

Chloroform: isoamyl alcohol (49: 1, v/v) Ethanol, Isopropanol, Liquid nitrogen, Phenol, Phosphate-buffered saline (PBS)

Required for cells grown in suspension and monolayers only.

Sodium acetate (2 M, pH 4.0)

Solution 0 (denaturing solution)

4 M guanidinium thiocyanate

25 mM sodium citrate'2HzO

0.5% (w/v) sodium lauryl sarcosinate

0.1Mp-mercaptoethanol

Dissolve250 g of guanidinium thiocyanate in 293 ml of H2O, 17.6ml of 0.75M sodium citrate (pH 7.0),

and 26.4 ml of 10%(w/v) sodium lauryl sarcosinate. Add a magnetic bar and stir the solution on a combination heater-stirrer at 65°C until all ingredients are dissolved. Store Solution D at room temperature, and add 0.36 ml of 14.4M stock p-mercaptoethanol per 50 ml of Solution D just before use. Solution D may be stored for months at room temperature but is sensitive to light. Note that guanidinium will precipitate at low temperatures.

Table 7-1 presents the amounts of Solution D required to extract RNA from various sources.

••. WARNING Solution 0 is very caustic. Wear appropriate gloves, a laboratory coat, and eye protection when preparing, handling, or working with the solution.

The amounts of Solution D recommended here are greater than those used by Chomczynski and Sacchi (1987). OUT experience and that of other investigators (e.g., Zolfaghari et al. 1993; Sparmann et al. 1997) indicate that the technique is more reproducible and the yield of RNA is consistently higher when the amount of solution 0 is increased to the values shown in the Table.

Stabilized formamide (Optional)

Stabilized formamide is used for the storage of RNA, please see the panel on STORAGE OF RNA following

Step II.

Cells and Tissues

Cells or tissue samples for RNA isolation

Centrifuges and Rotors

Sorvall SS-34 rotor or equivalent

Sorvall H1000 rotor or equivalent

Special Equipment

Cuvettes for measuring absorbance at 260 nm

The cuvettes should be either disposable UV-transparent methylacrylate or quartz. Before and after use, soak quartz cuvettes in concentrated HCI:methanol (I: I, v/v) for at least 30 minutes and then wash them extensively in sterile H20.

Homogenizer (e.g., Tissumizer from Tekmar-Dohrmann or Polytron from Brinkmann)

Mortar and pestle washed in DEPC-treated H20, prechilled.

Polypropylene snap-cap tube (e.g., Falcon) Water bath preset to 65°C Optional, please see Step 10.

METHOD

1. Prepare cells or tissue samples for isolation of RNA as appropriate for the material under study.

FOR TISSUES

When working with tissues such as pancreas or gut that are rich in degradative enzymes, it is best to cut the dissected tissue into small pieces (100 mg) and then drop the fragments immediately into liquid nitrogen. Fragments of snap-frozen tissue can be transferred to -70°C for storage or used immediately for extraction of RNA as described below. Tissues can be stored at -70°C for several months without affecting the yield or integrity of the RNA. Snap-freezing and pulverization is not always necessary. Tissues that are not as rich in RNases may be rapidly minced into small pieces and transferred directly into polypropylene snap-cap tubes

containing the appropriate amount of Solution D (Step c) below.

a. Isolate the desired tissues by dissection and place them immediately in liquid nitrogen.

b. Transfer -100 mg of the frozen tissue to a mortar containing liquid nitrogen and pulverize the tissue using a pestle. The tissue can be kept frozen during pulverization by the addition of liquid nitrogen.

c. Transfer the powdered tissue to a polypropylene snap-cap tube containing 3 ml of

Solution D.

d. Homogenize the tissue for 15-30 seconds at room temperature with a polytron homogemzer. Instead of grinding in a mortar, frozen tissue may be placed inside a homemade bag of plastic film and pulverized with a blunt instrument (e.g., a hammer) (Gramza et a!. 1995). Only certain types of plastic mOl are tough enough to withstand hammering at low temperature (e.g., Write-On Transparency Film from 3M).

FOR MAMMALIAN CElLS GROWN IN SUSPENSION

a. Harvest the cells by centrifugation at 200-1900g (1000-3000 rpm in a Sorvall RT600 using the HIOOO rotor) for 5-10 minutes at room temperature in a bench top centrifuge.

b. Remove the medium by aspiration and resuspend the cell pellets in 1-2 ml of sterile ice cold PBS.

c. Harvest the cells by centrifugation, remove the PBS completely by aspiration, and add 2ml of Solution D per 106 cells.

d. Homogenize the cells with a polytron homogenizer for 15-30 seconds at room temperature.

FOR MAMMALIAN CElLS GROWN IN MONOLAYERS

a. Remove the medium and rinse the cells once with 5-10 ml of sterile ice-cold PBS.

b. Remove PBS and lyse the cells in 2 ml of Solution D per 90-mm culture dish (1 ml per 60mm dish).

c. Transfer the cellly sates to a polypropylene snap-cap tube.

d. Homogenize the lysates with a polytron homogenizer for 15-30 seconds at room temperature.

2. Transfer the homogenate to a fresh polypropylene tube and sequentially add 0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of phenol, and 0.2 ml of chloroform-isoamyl alcohol per milliliter of Solution D. After addition of each reagent, cap the tube and mix the contents thoroughly by inversion.

3. Vortex the homogenate vigorously for 10 seconds. Incubate the tube for 15 minutes on ice to permit complete dissociation of nucleoprotein complexes.

4. Centrifuge the tube at 10,000g (9000 rpm in a Sorvall SS-34 rotor) for 20 minutes at 4°C, and then transfer the upper aqueous phase containing the extracted RNA to a fresh tube. To minimize contamination by DNA trapped at the interface, avoid taking the lowest part of the aqueous phase.

5. Add an equal volume of isopropanol to the extracted RNA. Mix the solution well and allow the RNA to precipitate for 1 hour or more at -20°C.

6. Collect the precipitated RNA by centrifugation at 10,000g (9000 rpm in a Sorvall SS-34 rotor) for 30 minutes at 4°C.

7. Carefully decant the isopropanol and dissolve the RNA pellet in 0.3 ml of Solution D for every 1 ml of this solution used in Step 1.

.••. IMPORTANT Pellets are easily lost. Decant the supernatant into a fresh tube. Do not discard it until the pellet has been checked.

8. Transfer the solution to a microfuge tube, vortex it well, and precipitate the RNA with 1 volume of isopropanol for 1 hour or more at -20°C. If degradation of RNA turns out to be a problem (e.g., when isolating RNA from cells or tissues known to contain large amounts of RNase, such as macrophages, pancreas, and small intestine), repeat Steps 7 and 8 once more.

9. Collect the precipitated RNA by centrifugation at maximum speed for 10 minutes at 4°C in a microfuge. Wash the pellet twice with 75% ethanol, centrifuge again, and remove any remaining ethanol with a disposable pipette tip. Store the open tube on the bench for a few minutes to allow the ethanol to evaporate. Do not allow the pellet to dry completely.

10. Add 50-100 ~l of DEPC-treated Hp. Store the RNA solution at -70°e. Addition of SDS to 0.5% followed by heating to 65°C may assist dissolution of the pellet.

11. Estimate the concentration of the RNA by measuring the absorbance at 260 nm of an aliquot of the final preparation, as described in Appendix 8. Purified RNA is not immune to degradation by RNase after resuspension in the 0.5% SDS solution.

Some investigators therefore prefer to dissolve the pellet of RNA in 50-100 fll of stabilized formamide and store the solution at -20°C (Chomczynski 1992). RNA can be recovered from formamide by precipitation with 4 volumes of ethanol. For further details, please see the panel on

STORAGE OF RNA.

SDS should be removed by chloroform extraction and ethanol precipitation before enzymatic treatment of the RNA (e.g., primer extension, reverse transcription, and in vitro translation). The redissolved RNA can then be used for mRNA purification by oligo(dT)-celiulose chromatography (Protocol 3) or analyzed by standard techniques such as blot hybridization (Protocols 7 and 8) or mapping (Protocols 10, 11, and 12).

RNA prepared from tissues is generally not contaminated to a significant extent with DNA. However, RNA prepared from cell lines undergoing spontaneous or induced apoptosis is often contaminated with fragments of degraded genomic DNA. RNA prepared from transfected cells is almost always contaminated by fragments of the DNA used for transfection. Some investigators therefore treat the final RNA preparation with RNase-free DNase (Grillo and Margolis 1990: Simms et al. 1993). Alternatively, fragments of DNA may be removed by preparing poly(A)+ RNA

by oligo(dT) chromatography.

STORAGE OF RNA

After precipitation with ethanol, store the RNA as follows:

• Dissolve the precipitate in deionized formamide and store at -2rY'C (Chomczynski 1992). Formamide provides a chemically stable environment that also protects RNA against degradation by RNases. Purified, salt-free RNA dissolves quickly in formamide up to a concentration of 4 mglml. At such concentrations, samples of the RNA can be analyzed directly by gel electrophoresis, RT-PCR, or RNase protection, saving time and avoiding potential degradation. If necessary, RNA can be recovered

from formamide by precipitation with 4 volumes of ethanol as described by Chomczynski (1992) or by diluting the formamide fourfold with 0.2 M NaCi and then adding the conventional 2 volumes of ethanol (Nadin-Davis and MezI1982).

• Dissolve the precipitate in an aqueous buffer and store at ~rY'c.Buffers commonly used for this purpose include SOS (0.1-{).50/0)in TE (pH 7.6) or in OEPC-treated H20 containing 0.1 mM EOTA(pH 7.5). The SOS should be removed by chloroform extraction and ethanol precipitation before enzymatic treatment of the RNA (e.g., primer extension, reverse transcription, and in vitro translation).

• Store the precipitate of RNA as a suspension at -2rY'C in ethanol. Samples of the RNA can be, as needed, with an automatic pipetting device. However, because precipitates of RNA are lumpy and sticky, and partly because of losses onto the surfaces of disposable pipette tips, the recovery of RNA is inconsistent.

This protocol exist in "Molecular Cloning" Sambrook and Russell book. Vol 1 chapter 7

Thank you Behzad that was very useful information

commercial RNA extraction kits from different companies will working well

Use Trizol.