Introduction. Principle. Storage

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1 Cntents Intrductin Principle Strage Kit Cntents Befre Starting Hmgenizatin f Samples Disruptin f sample with Mrtar-Pestle Disruptin and hmgenizatin with Rtr-Statr Hmgenizers Disruptin and hmgenizatin with Bead mills Hmgenizatin with syringe needle Ttal RNA/DNA Islatin Prtcl A. Islating RNA/DNA frm Animal Cells B. Islating RNA/DNA frm Animal Tissue Quantitatin and Strage RNA Quality Trubleshting Tips Revised June 2008 Intrductin E.Z.N.A. DNA/RNA Kit prvides a rapid and easy methd fr the islatin f ttal RNA and genmic DNA frm cultured cells and animal tissues. The kit allws single r simultaneus prcessing f multiple samples in less than 40 7 minutes. Nrmally, 1 x 10-1 x 10 eukarytic cells r mg f tissue can be used in a single experiment. There is n need fr phenl/chlrfrm extractins, and time-cnsuming steps such as CsCl gradient ultracentrifugatin, and precipitatin with isprpanl r LiCl, are eliminated. RNA purified using the E.Z.N.A.. DNA/RNA methd is ready fr applicatins + such as RT-PCR*, Nrthern bltting, ply A RNA (mrna) purificatin, nuclease prtectin, and in vitr translatin. Principle The E.Z.N.A.. DNA/RNA Kit cmbines reversible binding prperties f HiBind RNA technlgy with a specially designed buffer system which bind DNA t a DNA clumn befre RNA islatin. Samples are first lysed and hmgenized in a specially designed denature buffer (GTC), which immediately inhabits the activity f RNase and DNase. The lyste is then passed thrugh a HiBind DNA clumn which will selectively bind genmic DNA. After tw quick wash steps, the purified DNA is eluted frm HiBind DNA clumn. The flw-thrugh lysate frm the HiBind DNA clumn is then added with ethanl t create prper RNA binding cnditins, the sample is then laded int the HiBind RNA clumn t bind RNA. After tw wash steps, purified RNA is eluted with RNase-free water. Strage All cmpnents in the E.Z.N.A DNA/RNA Kit shuld be stred at rm temperature. During shipping and strage, crystals may frm in the GTC Lysis Buffer, simply warm t 37 C t disslve. All kit cmpnents are guaranteed fr at least 12 mnths frm the date f purchase. *The PCR prcess is cvered by U.S. Patents 4,83,195 and 4,83,202 (and internatinal equivalents) wned by Hffmann-LaRche, Inc. 2

2 Kit Cntents RNA Prep 5 RNA Prep 50 RNA Prep 200 Prduct Number R R R Purificatins Cmpnents HiBind RNA Clumns HiBind DNA Clumns ml Cllectin Tubes GTC Lysis Buffer 5 ml 40 ml 150 ml RNA Wash Buffer I 5 ml 30 ml 125 ml RNA Wash Buffer II 2 ml 12 ml 50 ml HB Buffer 5 ml 30 ml 125 ml DNA Wash Buffer 1.5 ml 15 ml 3 x 25 ml DEPC-ddH2O 1.0 ml 10 ml 40 ml Elutin Buffer 1.0 ml 5 ml 20 ml Instructin Manual 1 1 1! Under cl ambient cnditins, crystals may frm in GTC Lysis Buffer. This is nrmal and the bttle shuld be warmed t redisslve the salt.! 2-mercaptethanl (ß-mercaptethanl) is key in denaturing RNase and must be added t an aliqut f GTC Lysis Buffer befre use. Add 20 ìl f 2-mercaptethanl per 1 ml f GTC Lysis Buffer. This mixture can be stred fr 1 week at rm temperature.! All centrifugatin steps must be carried ut at 22 C-25 C. Disruptin and Hmgenizatin f samples Efficient disruptin and hmgenizatin f the sample is essential fr successfully islating ttal RNA. Cmplete disruptin f the cell walls and plasma membrane is very imprtant fr releasing all the RNA cntained in the samples. The purpse f hmgenizatin is t reduce the viscsity f the cell lysates prduced by cell disruptin. Incmplete hmgenizatin will reduce the binding f RNA t the RNA clumn and smetimes will clg the RNA clumn thus causing lwer yield r n yield. Disruptin f Sample with Mrtar and Pestle Befre Starting IMPORTANT RNA Wash Buffer II must be diluted with abslute ethanl befre use. R R R Add 8 ml 100% ethanl Add 48 ml 100% ethanl Add 200 ml 100% ethanl DNA Wash Buffer must be diluted with abslute ethanl befre use. R R R Add ml 100% ethanl Add 0 ml 100% ethanl Add 100 ml 100% ethanl Wear glves and take great care when wrking with liquid nitrgen. Excise tissue and prmptly freeze in a small vlume f liquid nitrgen. Grind tissue with a ceramic mrtar and pestle under apprximately 10 ml f liquid nitrgen and pur the suspensin int a pre-cled 15 ml plyprpylene Unless the tube is precled (in liquid nitrgen), the suspensin will bil vigrusly pssibly causing lss f tissue. When the liquid nitrgen has cmpletely evaprated, add GTC Lysis Buffer and cntinue with the prcedure as utlined belw. After interruptin f tissue, lysate can be hmgenized with Hmgenizer Spin Clumn (Prduct # HCR 002). The lysate is laded nt Hmgenizer Spin Clumn in a 2 ml cllectin Spin tw minutes at maximum speed in a micr centrifuge and then the hmgenized lysate is cllected. Use the Omega Hmgenizer Spin Clumn it is a fast and efficient way t hmgenize the lysate withut crss cntaminatin f samples. The alternate way t hmgenize the lysate is t use the syringe and needle. High mlecular-weight DNA can be sheared by passing the lysate thrugh a narrw needle (19-21 gauge) times.. Please take a few minutes t read this bklet thrughly and becme familiar with the prtcl. Prepare all materials required befre starting t minimize RNA degradatin.! Whenever wrking with RNA, always wear latex glves t minimize RNase cntaminatin. Use nly clean RNase-free dispsable plastic pipette tips when using the supplied reagents.! During the prcedure wrk carefully but quickly. Disruptin & hmgenizatin f sample with Rtr-Statr Hmgenizers Rtr-statr hm genizers can effectively sim ultaneusly disrupt and hmgenize mst samples. The prcess usually takes less than a minute depending n the tissue. Many Rtr-statr hm genizers perate with differently sized prbes r generatrs that allw prcessing sam ples in 50ml tubes. 4

3 Disruptin & hmgenizatin f sample using Bead Milling By using bead milling, cells and tissues can be disrupted and hmgenized by rapid agitatin in the presence f beads and lysis buffer. The ptimal use fr RNA islatin are 0.5mm glass beads fr yeast and unicellular cells, 4-8 mm beads fr animal tissue samples. Hmgenizatin f lysate with Syringe Needle Methd High m lecular weight DNA is respnsible fr the viscsity f cell lysates and can be shredded by passing the sample times thrugh a narrw needle (19-21 gauge). E.Z.N.A DNA/RNA Islatin Prtcl A. Islating Ttal RNA/DNA frm Animal Cells Materials supplied by user:! 2-mercaptethanl! Micrcentrifuge capable f at least 14,000 x g! 70% ethanl in DEPC-treated sterile distilled water! Sterile RNase-free pipette tips and 1.5ml centrifuge tubes! Dispsable latex glves Prcedure: 1.Determine the prper amunt f starting material: It is critical t use the crrect number f cells t btain ptimal RNA yield and purity. The maximum number f cells that can be prcessed with this DNA/RNA prtcl depends n: 1). the specific RNA cntents and type f cell lines; 2). The amunt f GTC Buffer required fr efficient lysis and maximum lading vlume f the Hibind RNA clumns; 3). The DNA capacity f the Hibind DNA clumns. The maximum binding capacity f the HiBind RNA clumn is 100ìg and the maximum DNA capacity fr HiBind DNA clumn is 20ìg. The maximum 7 number f the cells that GTC lysis buffer used in the this prtcl is 1 x 10. Use the fllwing table as a guideline t select a crrect starting material. Average Yield f Ttal cellar RNA Surce Number f Cells RNA Yield (ìg) IC21 1 x Hela 1 x HEK 1 x Harvest Cells: Fr cells grwn in suspensin: determine the number f cells. Pellet the apprpriate number f cells by centrifuge at 500 x fr 5 minutes. Aspirate the supernatant and cntinue step 3 f this prtcl. Fr cells grwn in a mnlayer: Cells grwn in a mnlayer in a cell culture dish can be directly lysed in the dish r trypsinized and then cllect the cell pellet befre lysis. Cells grwn in a cell culture flask shuld be trypsinized and cell pellet cllected prir t lysis Disrupt cells (d nt use mre than 1 x 10 cells) with GTC Lysis Buffer: Fr pelleted cells, lsen the cell pellet by flicking the tube and then add the apprpriate amunt f GTC Lysis Buffer based n the table belw. T directly lyse the cell in the culture dish, add the apprpriate amunt f GTC Lysis Buffer directly t the dish. Remember t add 20 ìl f 2-mercaptethanl per 1 ml f GTC Lysis Buffer befre use. GTC Lysis Buffer Vlume fr RNA Clumn Number f Cells Amunt f GTC Lysis Buffer (ìl) < 5 x x 10-1 x Hmgenize cells by using ne f the methds described n page 4-5. Nte: incmplete hmgenizatin f the sample will cause lwer yields and clgging f the clumn. It is recmmended t hmgenize the sample with rtr-statr hmgenizers since it nrmally prduces better yield. 5. Transfer the hmgenized lysate t a HiBind DNA clumn placed in a 2ml cllectin Centrifuge at x g fr 1 minute. Remve and save the clumn fr later DNA islatin (steps 11-15). Use the flw-thrugh fr the RNA islatin in the next step. Nte: make sure that all the liquid has passed thrugh the clumn after the centrifugatin. If necessary, repeat the centrifugatin until all liquid has passed thrugh the membrane. HIN3T3 1 x 10 15

4 RNA Islatin. Add an equal vlume f (350ìl r 700ìl) 70% ethanl t the flwthrugh and mix thrughly by pipetting up and dwn 20 times. If the sample lst vlume during hmgenizatin, adjust the vlume f the ethanl accrdingly. 7. Apply sample nt HiBind RNA spin clumn. The maximum capacity f the spin clumn is 800 ìl. (Larger vlumes can be laded successively.) A precipitate may frm in additin t the ethanl. Vrtex and add the entire mixture t the clumn. With the spin clumn inside the 2ml cllecting tube (supplied with kit), centrifuge at 10,000 x g fr 1 min at rm temperature. Discard flw-thrugh and prceed t next step.. Add 500ìl RNA Wash Buffer I directly int the HiBind RNA spin clumn. Centrifuge at 10,000 x g fr 1 minute t wash the clumn membrane. Discard flw-thrugh and re-use the cllectin 7. Place clumn int cllectin tube, and add 500ìl RNA Wash Buffer II t the clumn. Centrifuge at 10,000 x g fr 1 minute at rm temperature. Discard the flw-thrugh and cllectin Nte: Wash Buffer II Cncentrate must be diluted with abslute ethanl befre use. Refer t label n bttle fr directins. 8. Place the HiBind RNA spin clumn int a new cllectin Add 500ìl RNA Wash Buffer II directly int the HiBind RNA spin clumn. Centrifuge at 10,000 x g fr 1 minute t wash the spin clumn membrane. Discard the flw-thrugh and re-use the cllectin 9. Then with the cllectin tube emptied, centrifuge the HiBind RNA spin clumn fr 2 minutes at maximum speed t cmpletely dry the HiBind matrix Elutin f RNA. Transfer the clumn t a clean 1.5 ml centrifuge tube (Nt supplied) and elute the RNA with 40-70ìl f DEPC-treated water (supplied with kit). Make sure t add water directly nt clumn matrix. Centrifuge 1 min at 10,000 x g. A secnd elutin may be necessary if the expected yield f RNA is >30 ìg. Alternatively, RNA may be eluted with a greater vlume f water. While additinal elutins increase ttal RNA yield, the cncentratin will be lwered since mre than 80% f RNA is recvered with the first elutin. Pre-heat the water t 70 C befre adding t clumn and incubating clumn fr 5 min at rm temperature befre centrifugatin DNA Islatin may increase yields. 11. Place the DNA Binding clumn (frm step 5) int a new 2ml cllectin 12. Add 500ìl HB Buffer t the clumn and centrifuge at 10,000 x g fr 1 minute. Discard the flw-thrugh and re-use the cllectin 13. Add 700ìl f DNA wash Buffer t the DNA Binding clumn. Centrifuge at 10,000 x g fr 30 secnds. Discard the flw-thrugh and re-use the cllectin 14. Place the empty HiBind DNA clumn int a same cllectin Open the lid f the clumn and centrifuge at full speed fr 2 minutes t cmpletely dry the membrane. 15. Place the HiBind DNA clumn int a new 1.5 ml centrifuge tube (nt supplied). Add ìl Elutin Buffer directly t the center f the clumn membrane. Clse the lids and centrifuge at maximum speed fr 2 minutes t elute DNA. B. Islating Ttal RNA/DNA frm Animal Tissues Materials supplied by user:! 2-mercaptethanl! Micrcentrifuge capable f at least 14,000 x g! 70% ethanl in DEPC-treated sterile distilled water! Sterile RNase-free pipette tips and 1.5ml centrifuge tubes! Dispsable latex glves 1. Determine the prper amunt f starting material: This is critical t use the crrect number f cells t btain ptimal RNA yield and purity. The maximum number f cells that can be prcessed with this DNA/RNA prtcl varies depends n: 1). the specific RNA cntents and type f cell lines; 2). The amunt f GTC Buffer required fr efficient lysis and maximum lading vlume f the Hibind RNA clumns; 3). The DNA capacity f the Hibind DNA clumns. The maximum binding capacity f the HiBind RNA clumn is 100ìg and the maximum DNA remval capacity fr HiBind DNA clumn is 20ìg. The maximum amunt f tissue that GTC lysis buffer used in the this prtcl is 30mg. Use fllwing table as a guideline t select crrect starting material. If yu have n infrmatin abut yur starting material, use 10 mg as starting amunt, base n the 8

5 yield and quality f RNA btained frm 10 mg, adjust the starting amunt in the next purificatin. Average Yield f Ttal cellar RNA Surce Amunt f Tissue (mg) RNA Yield (ìg) Muse Tissue Brain Kidney Liver Heart 10 5 Spleen Lung Pancreas Thymus Disrupt Tissue and hmgenize the lysate in GTC Lysis Buffer using ne f the described methds n page 4. (D nt use mre than 30mg f tissue). Remember t add 20 ìl f 2-mercaptethanl per 1 ml f GTC Lysis Buffer befre use. Nte: incmplete hmgenizatin f the sample will cause lwer yields and clgging f the clumn. It is recmmended t hmgenize the sample with rtr-statr hmgenizers since it nrmally prduces better yield. GTC Lysis Buffer Vlume fr RNA Clumn Amunt f Tissue (mg) Amunt f GTC Lysis Buffer (ìl) Centrifuge at 13,000 x g fr 5 minutes. Carefully transfer the cleared supernatant t a HiBind DNA clumn pre-inserted in a 2ml cllectin Nte: In sme preparatins, a fatty upper layer will frm after the centrifugatin, transferring f any pellet r fatty layer may reduce the RNA yield r clg the clumn. 4. Centrifuge at x g fr 1 minute. Remve and save the HiBind DNA clumn fr later DNA islatin. Nte: make sure that all the liquid has passed thrugh the clumn after centrifugatin. If necessary, repeat the centrifugatin until all the liquid has passed thrugh the membrane. RNA Islatin 5. Add an equal vlume f (350ìl r 700ìl) 70% ethanl t the flwthrugh and mix thrughly by pipetting. If the sample lst vlume during hmgenizatin, adjust the vlume f ethanl accrdingly.. Apply sample nt HiBind RNA spin clumn. The maximum capacity f the spin clumn is 800ìl. (Larger vlumes can be laded successively.) A precipitate may frm in additin t the ethanl. Vrtex and add the entire mixture t the clumn. With the spin clumn inside the cllectin tube (supplied with kit), centrifuge at 10,000 x g fr 1 min at rm temperature. Discard flw-thrugh and re-use the cllectin 7. Add 500ìl RNA Wash Buffer I directly int the HiBind RNA spin clumn. Centrifuge at 10,000 x g fr 1 minute t wash the spin clumn membrane. Discard the flw-thrugh and re-use the cllectin 8. Place the clumn int a cllectin tube, and add 500ìl RNA Wash Buffer II t the clumn. Centrifuge at 10,000 x g fr 1 minute at rm temperature. Discard the flw-thrugh and cllectin Nte: Wash Buffer II Cncentrate must be diluted with abslute ethanl befre use. Refer t the label n the bttle fr directins. 9. Place the HiBind RNA spin clumn int a new cllectin Add 500ìl RNA Wash Buffer II directly int the HiBind RNA spin clumn. Centrifuge at 10,000 x g fr 1 minute t wash the spin clumn membrane. Discard flw-thrugh and re-use the cllectin 10. Then with the cllectin tube emptied, centrifuge the spin cartridge fr 2 minutes at maximum speed t cmpletely dry the HiBind matrix. 11. Elutin f RNA. Transfer the clumn t a clean 1.5 ml centrifuge tube (Nt supplied) and elute the RNA with 40-70ìl f DEPC-treated 10

6 DNA Islatin water (supplied with kit). Make sure t add water directly nt clumn matrix. Centrifuge 1 min at 10,000 x g. A secnd elutin may be necessary if the expected yield f RNA is >30 ìg. 12. Place the DNA Binding clumn (frm step 4) int a new 2ml cllectin 13. Add 500ìl HB Buffer t the clumn and centrifuge at 10,000 x g fr 1 minute. Discard the flw-thrugh and re-use the cllectin 14. Add 700ìl f DNA wash Buffer t the DNA Binding clumn. Centrifuge at 10,000 x g fr 30 secnds. Discard the flw-thrugh and re-use the cllectin 15. Place the empty clumn int a same cllectin Open the lid f the clumn and centrifuge at full speed fr 2 minutes t cmpletely dry the membrane. 1. Place the DNA Binding clumn int a new 1.5 ml centrifuge tube (nt supplied). Add ìl Elutin Buffer directly t the center f the clumn membrane. Clse the lids and centrifuge at maximum speed fr 2 minutes t elute DNA. Quantitatin and Strage f RNA T determ ine the cncentratin and purity f RNA, m easure absrbance at 20 nm and 280 nm in a spectrphtmeter. 1 O.D. unit measured at 20 nm crrespnds t 40 ìg f RNA per ml. DEPC-water is slightly acidic and can dram atically lwer absrbance values. W e suggest that yu dilute the sam ple in a buffered slutin (TE) fr spectrphtm etric analysis. The rati f A 20/A 280 f pure nucleic acids is 2.0, while fr pure prtein it is apprxim ately 0.. A rati f crrespnds t 90%-100% pure nucleic acid. (Phenl has an absrbance maximum at 275 nm and can interfere with absrbance readings f DNA r RNA. Stre RNA sam ples at -70 C in water. Under such cnditins RNA prepared with the E.Z.N.A. system is stable fr mre than a year. RNA Quality It is highly recmmended that RNA quality be determined prir t all analyses. The quality f RNA can best be assessed by denaturing agarse gel electrphresis and ethidium brm ide staining. Tw sharp bands shuld appear n the gel. These are the 28S and 18S (23S and 1S fr bacteria) ribsmal RNA bands. If these band smear twards lwer mlecular weight RNAs, then the RNA has undergne m ajr degradatin during preparatin, handling, r strage. Althugh RNA m lecules less than 200 bases in length d nt efficiently bind the HiBind matrix, a third RNA band, the trna band, may be visible when a large number f cells are used. Trubleshting Tips Prblem Cause Suggestin Little r n RNA eluted Clgged clumn RNA rem ains n the clum n! Repeat elutin.! Pre-heat DEPC-water t 70 C prir t elutin.! Incubate clumn fr 10 min with water prir t centrifugatin. Clumn is verladed! Reduce the quantity f the starting material. Incmplete hm genizatin! Cmpletely hmgenize sam ple.! Increase centrifugatin time.! Reduce the amunt f the starting material Degraded RNA Surce! Freeze starting material quickly in liquid nitrgen.! D nt stre tissue cultured cells prir t extractin unless they are lysed first.! Fllw prtcl clsely, and wrk quickly. Prblem in dwnstream applicatins DNA cntaminatin Lw Abs ratis RNase cntaminatin! Ensure nt t intrduce RNase during the prcedure.! Check buffers fr RNase cntam inatin. Salt carry-ver during elutin HiBind DNA clumn is verladed RNA diluted in acidic buffer r water! Ensure W ash Buffer II Cncentrate has been diluted with 4 vlum es f 100% ethanl as indicated n bttle.! 1 X W ash Buffer II m ust be stred and used at rm tem perature.! Repeat wash with W ash Buffer II.! Reduce the amunt f starting m aterial.! Digest with RNase-free DNase and inactivate at 75 C fr 5 m in.! DEPC-treated water is acidic and can dramatically lwer Abs20 values. Use TE buffer t dilute RNA prir t spectrphtmetric analysis. 12