EZ EXTRACTION Solution (EZol)
Contents
1. I Fee KoA EWR BRD www genedragon com tw Genomics BioSci amp Tech EZ Extraction Solution Instruction Manual Version 1 August 2005 Cat No GN EZO 001 50ml Cat No GN EZO 002 200ml Store at2 8 C EZ EXTRACTION Solution EZol User s Manual Reagent required but not supplied 1 2 Isopropyl alcohol 3 4 RNase free water or 0 5 SDS solution To prepare RNase free water draw Chloroform 75 Ethanol in DEPC treated water water into RNase free glass bottles Add diethypyrocarbonate DEPC to 0 1 v v Let stand overnight and autoclave SDS solution must be prepared using DEPC treated autoclaved water Precautions for Preventing RNase Contamination RNase can be introduced accidentally into RNA preparation at any point in the isolation procedure through improper technique Because RNase activity is difficult to be inhibited it is essential to prevent its introduction The following guidelines should be observed when working with RNA Always wear disposable gloves Skin often contains bacteria that can contaminate an RNA preparation and be a source of RNases Practice good microbiological technique to prevent microbial contamination Use sterile disposable plasticware and automatic pipettors reserved for RNA work to prevent cross contamination with RNases from shared equipment For example a laboratory that is using RNA probes will likely be using RNase Aor T1 to reduce background on2. a pipette several times The amount of EZol added is based on the area of the culture dish 1 ml per 10cm and not on the number of cells present An insufficient amount of EZol may result in contamination of the isolated RNA with DNA c Cells Grown in Suspension Pellet cells by centrifugation Lyse cells in EZol by repetitive pipetting Use 1 ml of EZol per 5 10 X 10 of animal plant or yeast cells or per 1x 10 bacteria cells Washing cells before addition of EZol should be avoided as this increases the possibility of mRNA degradation Disruption of some yeast and bacterial cells may require the use of a homogenizer 2 OPTIONAL An additional isolation step may be required for samples with high content of proteins fat polysaccharides or extracellular material such as muscles fatty tissue and tuberous parts of plants 12 000 rpm for 10 minutes at 2 8 C The resulting pellet contains extracellular membranes polysaccharides and high molecular weight DNA pellet while the supernatant contains RNA In samples from fatty tissues an excess of fat collects as a upper layer which should be removed In each case transfer the cleared homogenate solution to a fresh tube and proceed with chloroform addition and phase separation as described 3 PHASE SEPARATION Incubate the homogenized samples for 5 minutes at 15 30 C to allow the complete dissociation of nucleoprotein complexes Add 0 2 ml of chloroform per 1 ml of Ezol Cap sample tubes
3. securely Shake tubes vigorously by hand for 15 seconds and incubate them at 15 30 C for 3 5 minutes Centrifuge the samples at not more than 12 000 rpm for 15 minutes at 2 8 C Following centrifugation the mixture separates into a lower phenol chloroform phase a cloudy white interphase and a colorless upper aqueous phase RNA remains exclusively in the upper aqueous phase The volume of the aqueous phase is about 60 of the volume of EZol used for homogenization 4 RNA PRECIPITATION Transfer the aqueous phase to a fresh tube and save the organic phase if isolation of DNA or protein is desired Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol Use 0 5 ml of isopropyl alcohol per 1 ml of EZol used for the initial homogenization Incubate samples for 10 minutes at 15 30 C and then centrifuge at 12 000 rmp for 10 minutes at 2 10 C to form a gel like pellet at the bottom of the tube 5 RNA WASH Remove the supernatant To wash the RNA pellet add at least 1 ml of 75 ethanol per 1 ml of EZol used for the initial homogenization Mix the sample by vortexing and centrifuge at not more than 7 500 rpm for 5 minutes at 2 8 C 6 REDISSOLVING THE RNA At the end of the procedure briefly dry the RNA pellet air dry or vacuum dry for 5 10 minutes Do not dry the RNA by centrifugation under vacuum It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility Partially dissolved
4. RNA samples have an A260 A280 ratio lt 1 6 Dissolve RNA in RNase free water or 0 5 SDS solution by passing the solution through a pipette tip several times and incubate at 55 60 C for 10 minutes do not use SDS solution when RNA will be used in subsequent enzymatic reactions RNA can also be dissolved in 100 formamide deionized and stored at 70 C TROUBLESHOOTING GUIDE 1 Expected yield of RNA per mg of tissues or 1x 10 cultured cells Liver and spleen 6 10ug Kidney 3 4ug Skeletal muscles and brain 1 1 5ug Placenta 1 4ug Epithelial cells 8 5 ug Fibroblasts 5 7 ug 2 Low yield Incomplete homogenization or lysis of samples Final RNA pellet incompletely redissolved 3 A260 A280 ratio lt 1 65 RNA sample was diluted in water instead of TE prior to spectrophotometric analysis Low ionic strength and low pH solutions increase absorbance at 280nm See Wilfinger W et al Biotechniques 22 474 481 and Fox D K 1998 Focus 20 2 p 37 Insufficient amount of EZol was used in sample homogenization Following homogenization samples did not be placed at room temperature for 5 minutes The aqueous phase was contaminated with the phenol phase Incomplete dissolution of the final RNA pellet 4 RNA degradation Tissues did not be immediately processed or frozen after removal from animals Samples used for isolation or the isolated RNA preparations were stored at 5 20 C instead of 60 70 C Cultured Cells were disp
5. ersed by trypsin digestion Aqueous solutions or tubes were not RNase free Formaldehyde used for agarose gel electrophoresis had a pH below 3 5 5 RNA contamination Samples homogenized in too less volume of EZol Samples used for the isolation contained organic solvents e g ethanol DMSO strong buffer or alkaline solution 6 Proteoglycan and polysaccharide contamination The following modification of the RNA precipitation removes these contamination compounds from the isolated RNA Add to the aqueous phase 0 25 ml of isopropyl alcohol followed by 0 25 ml of a high salt precipitation solution 0 8 M sodium citrate and 1 2 M NaCl per 1 ml of EZol used for the homogenization Mix the resulting solution centrifuge and proceed with the isolation as described in the protocol The modified precipitation effectively precipitates RNA while maintaining polysaccharides and proteoglycans in solution A combination of modified precipitation step with an additional centrifugation of the initial homogenate is required to isolate pure RNA from plant materials containing a very high level of polysaccharides Storage condition 2 8 C EZol has demonstrated stability of 12 months when store at RT However we recommend to store at 2 8 C for optimal performance Ordering information Cat No EZO 001 50ml Cat No EZO 002 200ml
6. filters and any nondisposable items such as automatic pipettors can be rich sources of RNase In the presence of EZol RNA is protected from RNase contamination Downstream sample handing requires that nondisposable glassware or plasticware is RNase free Glassware can be baked at 150 C for 4 hours and plasticware can be soaked for 10 minutes in 0 5M NaOH rinsed thoroughly with water and autoclaved INSTRUCTION FOR RNA ISOLATION Colorless aqueous phase containing RNA White interphase Contain DNA nitain DI and protein Red organic phase P Prepare cells or tissues Add EZol Separate phase Add chloroform Transfer the upper aqueous phase to a new tube Precipitate RNA Wash and solubilize RNA pellet Store at 70 C CAUTION When working with EZol wear gloves and eye protection shield safety goggles Avoid contacting with skin or clothing Handle in a chemical fume hood Avoid breathing vapor Note Unless otherwise stated the procedure is carried out at 15 30 C and EZol is stored at 2 8 C 1 HOMOGENIZATION a Tissues Homogenize tissue samples in 1 ml of EZol per 50 100 mg of tissue using a glass Teflon or power homogenizer Polytron or Tekmar s TISSUMIZER or equivalent The sample volume should not exceed 10 of the volume of EZol used for homogenization b Cells Grown in Monolayer Lyse cells directly in culture dish by adding 1 ml EZol to a 3 5 cm diameter dish and pass the cell lysate through
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