ABIOpure TM Total RNA (version 2.0)

Transcription

1 ABIOpure TM Ttal RNA (versin 2.0) Bld Extractin Handbk Cat N: M541RP50-B FOR RESEARCH USE ONLY

2 Table f Cntents Cntents Page Kit Cmpnents 3 Precautins 3 Stability & Strage 4 General Descriptin 4 Limitatins 5 Quality Cntrl 5 Technical Supprt 5 Features 5 Samples 6 Reagent Preparatin 6 Materials required but nt prvided 6 Prtcl 7 Trubleshting 10 Ordering Infrmatin 12 02

3 Kit Cmpnents Cmpnent M541RP50-B Strage Temp N. f preps 50 RBC Lysis Buffer 50 ml 4 C Buffer RB1* 15 ml Rm Temperature Buffer RBW* 13 ml Rm Temperature Buffer RNW* 6 ml Rm Temperature RNase-free water 15 ml Rm Temperature BIOpure TM filter (yellw) with cllectin tube) BIOpure TM clumn type W (blue ring) with cllectin tube) 50 pcs Rm Temperature 50 pcs Rm Temperature 1.5 ml cllectin tube 50 pcs Rm Temperature *Befre first use, add abslute ethanl (ACS grade r better) int buffers RB1, RBW, and RNW as indicated n the bttle Precautins RBC Lysis Buffer cntains phenl which is pisnus and quinidine salt which is an irritant. Care shuld be taken during handling. Always wear a lab cat, dispsable glves, prtective gggles and fllw standard safety precautins. In case f cntact, wash immediately with plenty at rm temperature water and seek medical advice. Handle and dispse f all bilgical samples as if they were able t transmit infective agents. Avid direct cntact with the bilgical samples. Avid prducing spills r aersl. Any material cming in cntact with the bilgical samples must be treated fr at least 30 minutes with 3% sdium hypchlrite r autclaved fr ne hur at 121 C befre dispsal. 03

4 Never pipette slutins by muth! Handle and dispse f all reagents and all materials used t carry ut the assay as if they were able t transmit infective agents. Avid direct cntact with the reagents. Avid prducing spills r aersl. Waste must be handled and dispsed f accrding t adequate safety measures. Dispsable cmbustible material must be incinerated. Stability and Strage ABIOpure TM Ttal RNA Bld Extractin kit, except the RBC Lysis Buffer, shuld be stred at rm temperature. RBC Lysis Buffer shuld be stred 4 C fr ptimal perfrmance. All cmpnents are stable fr 1 year. General Descriptin ABIOpure TM Ttal RNA Bld Extractin kit is designed fr the purificatin f ttal RNA frm fresh mammalian whle bld. This kit utilizes cell lysis and purificatin based n glassfiber membrane technlgy. Whle bld sample is hmgenized and lysed in the RBC Lysis Buffer, a mnphasic slutin cntaining phenl and guanidium salt, which rapidly lyses cells and inactivates the nucleases. In cnventinal methds, the erythrcytes f mammalian bld, which d nt cntain nuclei (nr RNA), shuld be remved by pretreatment such as smtic lysis. This type f pretreatment increases the experiment time and the pssibility f RNA-breakage, fllwed by decline f RNA quality. The ABIOpure TM Ttal RNA Bld Extractin kit des nt need additinal pretreatment as the bld is lysed in ne step. Then additin f chlrfrm effects a separatin f the lysate int aqueus and rganic phases. After phase-separating, DNA and prteins remain in the interphase and the rganic phase respectively, and the released RNA exists in the aqueus phase. The aqueus phase is picked and applied t an ABIOpure TM filter t eliminate small amunts f cntaminated DNA and ther bld cntaminants. The passed-thrugh is mixed with Buffer RB1, RNA binding buffer, and then the mixture is applied t a 04

5 mini spin clumn. After a series f washing with Buffers RBW and RNW, pure RNA can be eluted by RNase-free water. The entire prcedure can be cmpleted within 30 min and the purified RNA is ready fr use in the islatin f Ply A+ RNA, Nrthern bltting, dt bltting, in vitr translatin, clning, RT-PCR, PRA and ther analytical prcedures. Limitatins The ABIOpure TM Ttal RNA Bld Extractin kit is intended fr Research Use Only applicatins. Quality Cntrl All cmpnents f the ABIOpure TM Ttal RNA Bld Extractin kit are manufactured in a clean envirnment which is mnitred peridically. T ensure prduct cnsistency and quality, the quality certificatin prcess is carried ut n each lt f prduct. Technical Supprt If yu need assistance, have any questin r suggestin r if yu experience any difficulties using ABIOpure TM extractin kits, please feel free t cntact ur technical supprt team at supprt@alliancebi.cm. Features Frmat: Spin Clumn Maximum amunt f starting sample: 0.25 ml Minimum amunt f starting sample: 0.10 ml Maximum lading vlume: 700 µl Minimum elutin vlume: 30 µl Maximum binding capacity: 100 µg Typical yield: 3 µg Operatin time: 30 minutes 05

6 Samples Up t 250 µl f mammalian whle bld. Cllect bld samples in EDTA-Na 2 treated cllectin tubes (r ther anticagulant mixture that des nt affect yur later applicatin). Start the extractin prcedure within 4 hurs after cllectin r stre at 4 C fr maximum 12 hurs befre extractin. T ensure that the RNA extract cntains enugh distributin f mrnas, avid strage f samples mre than few hurs after islatin. Fr lng-term strage, it is recmmended t stre the sample lysate at -70 C (after prper mixing and hmgenizatin with Buffer RB and β-mercaptethanl). Materials required but nt prvided Laminar flw hd Pipette set (10 µl, 100 µl and 1000 µl) RNA-free pipet tips Sterile 1.5 micrcentrifuge tubes (nuclease-free) Micrcentrifuge fr centrifugatin at 4 0 C and at rm temperature Chlrfrm r I-brm-3-chlrprpane (BCP) Suitable prtectin (ex. lab cat, dispsable glves, gggles, etc.) 06

7 Mammalian Bld Prtcl Befre first use, add abslute ethanl (ACS grade r better) int buffers RB1, RBW, and RNW as indicated n the bttle. 1. Add 750 µl RBC Lysis Buffer in a 1.5 ml micrcentrifuge tube (nt prvided). Add 250 µl bld sample t the 1.5 ml micrcentrifuge tube and vrtex vigrusly. 2. If sample vlume is 100 µl, sample shuld be adjusted t 250 µl with PBS r RNase-free water. Be sure t cnfirm the applicable minimum vlume, which is 100 µl Incubate 2 minutes at rm temperature. This step allws leukcytes t be cmpletely cllapsed. Add 0.2 ml chlrfrm. Shake vigrusly fr 15 secnds and let stand 2 minutes at rm temperature. Alternatively, 0.1 ml BCP (I-brm-3-chlrprpane) can be used in place f chlrfrm. Centrifuge at 12,000 x g fr 15 minutes at 4 0 C. 5. The mixture will be separated int 3 phases: a lwer layer, an interphase, and a clrless upper aqueus layer. The upper aqueus vlume is apprx. 450 µl. Centrifugatin at > 8 0 C may cause sme DNA t partitin in the aqueus phase. 07

8 Transfer the aqueus phase (apprx. 450 µl) t the ABIOpure TM filter (yellw). Small amunt f DNA and ther bld cntaminants are eliminated by the ABIOpure TM filter. Centrifuge at > 10,000 x g fr 30 secnds at rm temperature. Add 2X vlume (usually 900 µl) f Buffer RB1 t the cllectin tube including the passed-thrugh, and mix well by pipetting. D nt centrifuge at this step. Transfer up t 700 µl f mixture t an ABIOpure TM clumn (blue ring). Centrifuge at > 10,000 x g fr 30 secnds at rm temperature Discard the passed-thrugh and reinsert the mini spin clumn back int the same tube. Repeat steps 9-10 using the remainder f the sample. Add 500 µl f Buffer RBW t the mini spin clumn. Centrifuge at > 10,000 x g fr 30 secnds at rm temperature. 13. Discard the passed-thrugh and reinsert the mini spin clumn back int the same tube. 14. Add 500 µl f Buffer RNW t the mini spin clumn. 08

9 Centrifuge at > 10,000 x g fr 30 secnds at rm temperature. 15. Discard the passed-thrugh and reinsert the mini spin clumn back int the same tube. Centrifuge at > 10,000 x g fr an additinal 1 minute at rm temperature t remve residual wash buffer Transfer the mini spin clumn t a new 1.5 ml micrcentrifuge tube (prvided). Residual ethanl may interfere with dwnstream reactins. Care must be taken at this step fr eliminating the carryver f Buffer RNW. Add 50 µl f RNase-free water t the center f the membrane in the mini spin clumn. T increase the RNA cncentratin, reduce the elutin vlume at least 30 µl. Centrifuge at 10,000 x g fr an additinal 1 minute at rm temperature. 18. Purified RNA can be stred at 4 C fr immediate analysis and can be stred at -70 C fr lng term strage. The purified RNA is free f DNA and prteins, and A260/A280 will be between 1.8 and

10 Trubleshting Prblem Pssible Causes Suggested Slutins Lw yield f RNA Pr quality f bld sample Prcess the fresh bld sample immediately. Sample nt cmpletely lysed Vrtex the sample vigrusly. Be sure t incubate fr 2 minutes at rm temperature after lysis step. T much r t less bld sample It may cause an inefficient lysis effect. Use the apprpriate sample vlume frm 100 µl t 250 µl. Sme aqueus phase left Perfrm 2 nd extractin with the remaining aqueus phase. Incrrect elutin cnditins Add RNase-free water t the center f the mini spin clumn membrane. Sample manipulated t much befre the additin f RBC Lysing Buffer Prcess the bld sample immediately after harvest Degradatin f RNA RNA still bund t the RB Clumn membrane Stre bld sample at C (nt recmmended). As strage time increases, RNA cnditin will deterirate. 10

11 Degradatin f RNA Reagent r dispsable is nt RNase-free Make sure t use nly RNase-free prducts Inapprpriate handling f starting material Ensure handling and strage f samples accrding t the prtcl RNase cntaminatin Avid wrking n areas where DNA preparatin is perfrmed with the presence f RNase. Lw A260/280 (<1.6) Hi DNA cntaminatin Aqueus phase was cntaminated with the phenl phase Avid carryver when transferring the aqueus phase t ABIOpure TM filter. Sample nt cmpletely lysed with RBC Lysing Buffer Use 750 µl RBC Lysing Buffer fr up t 250 µl bld sample. Be sure t incubate sample fr 2 minutes at rm temperature after lysis step. N treatment with DNase Perfrm ne r bth ptinal steps f DNA Residue Degradatin. 11

12 Ordering Infrmatin Prduct Name Cat. N. # f Preps ABIOpure TM Ttal DNA Bld/Tissue/Cell M501DP preps ABIOpure TM Ttal RNA Cell-Free Fluids M541RP50-A 50 preps ABIOpure TM Ttal RNA Bld M541RP50-B 50 preps ABIOpure TM Viral DNA/RNA M561VT50 50 preps rd Drive SE Suite 150 Bthell, WA USA T: F: Fr Technical Supprt: supprt@alliancebi.cm ABIOpure Viral_M561_v2_v11 FOR RESEARCH USE ONLY 2011 ALLIANCE BIO, all rights reserved 12