Omega公司RNA提取试剂盒说明书

RNA-Solv®

ReagentRNA Isolation SolventWARNING: This reagent is toxic if swallowed and in contactwith skin. Causes burns. After contact with skin, washimmediately with copious amounts of mild detergent andwater. If you feel sick, seek medical advice at once and t No:R6830-00 (5 ml) R6830-01 (100 ml) R6830-02 (200 ml)Storage Conditions: RNA-Solv®

is stable for at least 24 monthswhen stored at 2°C-8°C and yields reproducible results.

IntroductionRNA-Solv® Reagent is a reagent system for the isolation of totalRNA from cells and tissues. The reagent, a single-phase solutionconsisting of phenol and guanidine isothiocyanate, is modificationof the single-step RNA isolation method developed by Chomczynskiand Sacchi (1).The sample is homogenized and lysed in RNA-Solv® Reagent which maintains the integrity of the RNA, whiledisrupting and denaturing endogenous RNases and other cellularcomponents. Extraction of the lysate with chloroform furtherdenatures proteins and separates the mixture into an organic andan aqueous phase. RNA remains exclusively in the aqueous phase,and is subsequently recovered by isopropanol.

This method is suitable for small quantities of tissue (<100 mg) andcells (<5 X106), and large quantities of tissue ( up to1 g) and cells(<10

8 ), of human, animal, plant, or bacterial origin. The simplicityof the RNA-Solv® Reagent method allows simultaneous processingof a large number of samples. The entire procedure can becompleted in one hour. Total RNA prepared in this manner can beused for Northern blot analysis, dot blot hybridization, poly(A) +selection, in vitro translation, RNase protection assay, andmolecular cloning. For use in amplification by thermal cycling,treatment of the isolated RNA with RNase-free DNase I isrecommended when the two amplimers lie within a single ed By User•Chloroform (no isoamyl alcohol added)•Isopropyl alcohol•80% Ethanol (in DEPC-treated water)•RNase-free water•Tabletop centrifuge capable of 12,000 x g at room temperatureGeneral Notes Regarding RNase ContaminationWhenever working with RNA :•Always wear disposable gloves and change gloves frequently.•Use sterile, disposable plasticware and automatic pipettesreserved for RNA work to prevent cross-contamination withRNases.•In the presence of RNA-Solv® Reagent, RNA is protected fromRNase contamination. Downstream sample handling requiresthat nondisposable glassware or plasticware be RNase-free.•Use only DECP-treated buffers. Add DEPC to a finalconcentration of 0.1%, incubate at 37oC for 2 hours, andautoclave at 121oC. Do not add DEPC to Tris buffers. Suchbuffers must be prepared by using tionUse only disposable polypropylene tubes for small samples andglass Corex tubes for larger samples. All tubes must be able towithstand 12,000 x g . Polystyrene tubes may crack with chloroformBefore StartingA. Small Samples :To isolate RNA from very small samples (<106cells or <10 mg tissue) perform homogenization (or lysis) ofsamples in 0.8 mL of RNA-Solv®, and add 1 mg RNase-freeglycogen or yeast tRNA as carrier. This will improve yields obtainedwith precipitation.B. Difficult Animal Samples: Specimens containing large amountsof proteins, fat, polysaccharides or extracellular material such asmuscles, fat tissue, and sperm, will require the followingmodification. After lysis/homogenization in RNA-Solv® Reagent,centrifuge at 12,000 x g for 10 minutes at room temperature toremove insoluble debris. Often a precipitate forms at the bottom ofthe tube, but with fatty tissue, a lipid layer will also form above theaqueous phase. The supernatant will contain the RNA and must becarefully transferred to a fresh 1.5 ml microfuge tube beforeproceeding.C. Interruption the procedure: Following lysis in RNA-Solv®Reagent and before addition of chloroform, samples can be storedat -70oC for up to 3 months. In addition, once the RNA isprecipitated in isopropanol, the pellet may be stored at -20oC or -70oC for up to 1 year.

RNA-Solv® Protocol for Total RNA IsolationCAUTION: When working with RNA-Solv® Reagent use gloves andeye protection (safety goggles) and avoid contact with skin orclothing. Work in a chemical fume hood to avoid inhaling vapor.

Unless otherwise noted, all steps are to be carried out at roomtemperature (20oC-25oC).1. Homogenization and lysis of samples:

follow either methodbelowa) Tissue SamplesHomogenize tissue samples in 1 mL of RNA-Solv® Reagent per 50-100 mg of tissue using an appropriate mechanical homogenizer.

Alternatively one can pulverize tissue in liquid nitrogen with mortarand pestle and transfer the powder to a clean 1.5 ml microcentrifugetube. If ceramic mortar and pestle are not available, homogenize thesample in the microfuge tube using a disposable microtube pestle(Eppendorf, Cat No. 0030 120.973; VWR, Cat No. KT 749520-0000). The sample volume should not exceed 10% of the volumeof RNA-Solv® Reagent used.b) Cells Grown in SuspensionPellet cells by centrifugation. Lyse cells in RNA-Solv® Reagent byrepetitive pipetting. Use 1 mL of the reagent per 5-10 x 106 ofanimal, plant or yeast cells, or per 1 x 108 bacterial cells. Washingcells before addition of RNA-Solv® Reagent should be avoided asthis increases the possibility of mRNA degradation and RNasecontamination. For plant, fungal, and yeast cells mechanical orenzymatic homogenization may be required. Also, for plant, fungal,and yeast cells, we recommend the use of the E.Z.N.A.® Plant(R6627),Fungal (R6640), and Yeast (R6670) RNA Kits from OmegaBio-tek.c) Cells Grown in Monolayer

Lyse cells directly in a culture dish by adding 1 mL of RNA-Solv®Reagent to a 3.5 cm diameter dish, and passing the cell lysateseveral times through a blue pipette tip. The amount of RNA-Solv®Reagent added is based on the area of the culture dish (~1 mL per10 cm2 ). An insufficient amount of RNA-Solv® Reagent may resultin contamination of the isolated RNA with DNA. Always use moreRNA-Solv® Reagent if in the lysate is too viscous to aspirate witha pipette.2. Add 0.2 mL of chloroform per 1 mL of RNA-Solv® sample tubes securely and vortex vigorously for 15 te on ice for 10 minutes. This step is critical - do not changeit.3. Centrifuge the samples at no more than 12,000 x g for 15minutes 4EC. The mixture separates into a lower phenol-chloroform phase, an interphase, and an upper aqueous remains entirely in the aqueous phase.4. Precipitation of RNA. Transfer no more than 80% of theaqueous phase to a fresh tube, and discard the lower organicphase. Precipitate the RNA from the aqueous phase by adding 500ìl of isopropyl alcohol per 1 mL of RNA-Solv® Reagent used for theinitial homogenization. Incubate samples at room temperature 10minutes and centrifuge at no more than 12,000 x g for 10 minutesalso at room ydrate-rich samples: Plant samples of high polysaccharidecontent or animal tissues rich in glycosaminoglycans(proteoglycans) require the following modified precipitation methodfor obtaining pure RNA. Prepare Buffer A ( 1.2 M sodium chloride,800 mM sodium citrate). Following step 3, add to the aqueousphase 0.3 ml isopropanol followed by 0.3 ml Buffer A per 1 mlRNASolv ® Reagent used in step 1. Vortex to mix and centrifuge atno more than 12,000 x g for 10 minutes at room temperature. Thishigh salt precipitation will reduce co-purification of complexcarbohydrates.5. Wash RNA pellet. Discard the supernatant and wash the RNApellet once with 1 ml 80% ethanol. Mix the sample by vortexing andcentrifuge at no more than 7,500 x g for 5 minutes at roomtemperature.6. Reconstitute RNA. Carefully aspirate and discard the ethanoland briefly AIR DRY the RNA pellet for 2-5 minutes at roomtemperature. Do not use centrifugal devices equipped with avacuum source as over-drying will lead to difficulty in re-dissolvingRNA in water. Dissolve RNA in RNase-free water - a 5 minuteincubation at 60 °C may be required. RNA can also be reconstitutedin 100% formamide (deionized) and stored at -70°C.

RNA is now suitable for RNase protection, northern analysis andreverse transcriptase reactions. For isolation of poly(A)+ RNA anadditional ethanol precipitation is required. Add 1/8 X volume ofRNase-free 3M NaAc, pH 6.0 followed by 2.5 X volume absoluteethanol. Vortex to mix and incubate at room temperature for 5minutes. Centrifuge at 12,000 x g for 10 min at room temperatureand discard the supernatant. Wash the pellet as before andreconstitute in DECP-treated water.

Determination of Yield and QualityUV spectrophotometric analysis of the purified RNA is required forobtaining yield. To do so, dilute the RNA in an appropriate volumeof TE buffer, pH 8.0 (not water; RNA yields low Abs ratio values ifdissolved in acidic buffers) and measure absorbance at 260 nm andat 280 nm.

RNA Conc = 40 ìg/ml X Dilution factor X Abs 260 nmTypical Abs 260 nm/ 280 nm ratios of 1.7-1.9 are obtained with theprotocol. Yields vary depending of type and amount of startingmaterial, and on condition of storage prior to processing. Forassessing the quality of RNA, we recommend you performdenaturing agarose gel electrophoresis to confirm the integrity ofpurified material. Invariably, the full spectrum of RNAs, including4S and 5S species are purified with RNA-Solv® ed Yields per 1 mg tissue or 106 cells:Liver and spleen, 5-10 ìgKidney, 2-5 ìgBrain, 1-2 ìgEndothelial cells, 7-12 ìgFibroblasts, 6-8 ìgTroubleshooting•Low RNA Yields: Incomplete lysis of samples in RNASolvReagent. RNA pellet not completelt dissolved in DEPC-water.

pH of diluent used for spectrophotometric analysis is too low.•Degraded RNA: Tissues were not immediately processed orfrozen. Inadequate storage of starting material prio toisolation. Inadequate storage of RNA (-5 to -20°C, instead of-60 to -70°C) Trypsin/EDTA was used in dislodging monolayercells. Buffers or plasticwasre were not dehyde used for denaturing agarose-gelelectrophoresis had a pH below 3.0.•Low Abs260/Abs280 ratios: Sample was diluted in waterrather than TE. Acidic pH lowers absorbance ratios. Use TEbuffer as diluent for readings. Insufficient RNASolv Reagentwas used for lysis of sample. Ice incubation in step 2 was notperformed. The aqueous phase was contaminated with thephenolic phase.•DNA contamination of RNA: Too little RNASolv Reagent usedfor sample processing causing inadequate separation ofDNA/nucleoprotein complexes from aqueous RNA. Theaqueous phase was contaminated with the phenol cal Support:Omega Bio-tek, USA - call toll-free : 1 888 832 8896

References:

1. Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, 156

(1987).2. Chomczynski, P. Biotechniques 15, 532 (1993).For laboratory research use N: Not for diagnostic use. The safety and efficacy ofthis product indiagnostic or other clinical uses has not been ,1999 (C). All rights reserved by Omega Bio-tek, Inc.

RNA-Solv is a registered mark of Omega Bio-tek, Inc.