RY43 Purification of total RNA incl...sma using the
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1. 15 25 C for 5 min This step promotes dissociation of nucleoprotein complexes 4 Optional Add 5 pl of 5 nM Syn cel miR 39 miScript miRNA Mimic Details of how to prepare a 5 nM miScript miRNA Mimic working solution are provided in Things to do before starting 5 Add 1 volume chloroform to the tube containing the homogenate and close securely see table 1 for guidelines Vortex the tube vigorously for 15 s Thorough mixing is important for subsequent phase separation 6 Place the tube containing the homogenate on the benchtop at room temperature for 2 3 min Purification of RNA from serum or plasma RY43 Feb 11 page 3 of 6 10 11 12 13 Centrifuge for 15 min at 12 000 x g at 4 C After centrifugation heat the centrifuge up to room temperature 15 25 C if the same centrifuge will be used for the next centrifugation steps After centrifugation the sample separates into 3 phases an upper colorless aqueous phase containing RNA a white interphase and a lower red organic phase For the approximate volume of the aqueous phase see Table 1 Transfer the upper aqueous phase to a new collection tube supplied Avoid transfer of any interphase material Add 1 5 volumes of 100 ethanol and mix thoroughly by pipetting up and down several times Do not centrifuge Continue without delay with step 9 For the approximate volume of the aqueous phase and the volume of ethanol to add see Table 1 A precipi2. buffer BW Syn cel miR 39 miScript miRNA Mimic cat no MSY0000010 BE Method for C elegans miRNA detection e g miScript PCR System in combination with the Ce miR 39 1 miScript Primer Assay cat no MS00019789 Important points before starting If using the miRNeasy Mini Kit for the first time read Important Notes in the miRNeasy Mini Handbook If working with RNA for the first time read Appendix E in the miRNeasy Mini Handbook Qo 000906 QIAGEN Sample amp Assay Technologies Things After collection and centrifugation plasma or serum can be stored at 2 8 C for up to 6 hours or used directly in the procedure For long term storage freezing at 20 C or 80 C in aliquots is recommended To process frozen homogenized lysates incubate at 37 C in a water bath until samples are completely thawed and salts are dissolved Avoid prolonged incubation which may compromise RNA integrity DNase digestion is not recommended for plasma or serum samples The combined QlAzol and RNeasy technologies efficiently remove most of the trace amounts of DNA in plasma and serum In addition miScript Primer Assays and most other assays for mature miRNA are not affected by the presence of small amounts of genomic DNA In some cases on column DNase treatment may reduce recovery of small RNA from plasma or serum Buffer RWT may form a precipitate upon storage If necessary redissolve by warming and then place at room
3. temperature 15 25 C QIAzol Lysis Reagent and Buffer RWT contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See the Safety Information in the miRNeasy Mini Handbook Except for phase separation step 7 all protocol and centrifugation steps should be performed at room temperature The procedure is suitable for use with serum samples or with plasma samples containing either citrate or EDTA Plasma samples containing heparin should not be used because this anticoagulant can interfere with downstream assays such as RT PCR to do before starting Buffers RWT and RPE are supplied as concentrates Before using for the first time add the required volumes of ethanol 96 10096 as indicated on the bottle to obtain a working solution Optional If desired synthetic C elegans miRNA can be added to samples to control for variations during the preparation of total RNA and subsequent steps After purification real time RT PCR detection of the C elegans miRNA can be performed and these results can be used for normalization of real time RT PCR results of endogenous miRNAs in the sample This corrects for variations during RNA preparation cDNA synthesis and real time PCR We recommend Syn cel miR 39 miScript miRNA Mimic cat no MSYO000010 for this purpose as it shows no sequence homology to any known human mouse or rat miRNA miScript miRNA Mimics are provided lyophilized Prepare miScr
4. QIAGEN Supplementary Protocol Purification of total RNA including small RNAs from serum or plasma using the miRNeasy Mini Kit This protocol is intended as a guideline for the purification of total RNA including small RNAs e g miRNAs from serum and plasma using the miRNeasy Mini Kit cat no 217004 IMPORTANT Please read the miRNeasy Mini Handbook paying careful attention to the Safety Information and Important Notes sections before beginning these procedures Handbooks can be found at www qiagen com handbooks miRNeasy Kits are intended for molecular biology applications These products are not intended for the diagnosis prevention or treatment of a disease Equipment and reagents to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Chloroform without added isoamyl alcohol Ethanol 7096 and 96 10096 do not use denatured alcohol which contains other substances such as methanol and methylethylketone Sterile RNase free pipet tips 1 5 ml or 2 ml microcentrifuge tubes Microcentrifuge s with rotor for 2 ml tubes for centrifugation at 4 C and at room temperature 15 25 C Disposable gloves Optional if using C elegans miRNA for normalization and or as an internal control M RNase free TE
5. aly 800 787980 Sweden 020 790282 China 021 51345678 Japan 03 5547 0811 Switzerland 055 254 22 11 eoe Denmark 80 885945 Korea South 1544 7145 UK 01293 422 911 QI AGEN Finland 0800 914416 Luxembourg 8002 2076 USA 800 426 8157 Sample amp Assay Technologies
6. ipt miRNA Mimic stock and working solution as follows Briefly centrifuge the miScript miRNA Mimic tube prior to opening as some of the product may have been dislodged during shipping Resuspend with an appropriate volume of RNase free water provided to obtain a 1 UM stock solution For example resuspend 1 nmol miScript miRNA Mimic in 1 ml RNase free water To obtain a 5 nM working solution further dilute 5 ul of 1 uM stock solution in 995 ul RNase free TE Purification of RNA from serum or plasma RY43 Feb 11 page 2 of 6 buffer Aliquot stock and working solution and store at 20 C for future use Aliquots are stable for 18 months Procedure 1 Prepare serum or plasma or thaw frozen samples 2 Add 5 volumes QlAzol Lysis Reagent see table 1 for guidelines Mix by vortexing or pipetting up and down Table 1 Reagent volumes for various starting volumes of serum plasma Serum plasma ul Protocol step 2 Protocol step 4 Protocol step 8 Protocol step 8 QGlAzol Lysis Chloroform ul Approx volume of 100 Ethanol Reagent ul upper aqueous ul phase ul lt 50 250 50 150 225 100 500 100 300 450 200 1000 200 600 900 Note If the volume of plasma or serum is not limited we recommend using 100 200 ul per RNA preparation Note After addition of QlAzol Lysis Reagent lysates can be stored at 70 C for several months 3 Place the tube containing the homogenate on the benchtop at room temperature
7. ollection tube not supplied and discard the old collection tube with the flow through Centrifuge in a microcentrifuge at full speed for 2 min The long centrifugation step dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions 15 Transfer the RNeasy Mini spin column to a new 1 5 ml collection tube supplied Pipet 30 50 pul RNase free water directly onto the RNeasy Mini spin column membrane Close the lid gently and centrifuge for 1 min at 28000 x g 210 000 rpm to elute the RNA Purification of RNA from serum or plasma RY43 Feb 11 page 5 of 6 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor Material safety data sheets MSDS for any QIAGEN product can be downloaded from www giagen com Support MSDS aspx Trademarks QIAGEN QIAzol RNeasy QIAGEN Group RY43 Feb 11 2011 QIAGEN all rights reserved www giagen com France 01 60 920 930 The Netherlands 0800 0229592 Australia 1 800 243 800 Germany 02103 29 12000 Norway 800 18859 Austria 0800 281010 Hong Kong 800 933 965 Singapore 65 67775366 Belgium 0800 79612 Ireland 1800 555 049 Spain 91 630 7050 O eo C Canada 800 572 9613 It
8. tate may form after addition of ethanol but this will not affect the procedure Pipet up to 700 yl of the sample including any precipitate that may have formed into an RNeasy Mini spin column in a 2 ml collection tube both supplied Close the lid gently and centrifuge at 28000 x g 210 000 rpm for 15 s at room temperature 15 25 C Discard the flow through Reuse the collection tube in step 10 Repeat step 9 using the remainder of the sample Discard the flow through Reuse the collection tube in step 11 Add 700 ul Buffer RWT to the RNeasy Mini spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the column Discard the flow through Reuse the collection tube in step 12 Pipet 500 pl Buffer RPE onto the RNeasy Mini spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the column Discard the flow through Reuse the collection tube in step 13 Repeat step 12 Note Following centrifugation remove the RNeasy Mini spin column from the collection tube carefully so the column does not contact the flow through Otherwise carryover of ethanol will occur Flow through contains QIAzol Lysis Reagent or Buffer RWT and is therefore not compatible with bleach See the Safety Information in the miRNeasy Mini Handbook Purification of RNA from serum or plasma RY43 Feb 11 page 4 of 6 14 Place the RNeasy Mini spin column into a new 2 ml c
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