Kit for mirna isolation from animal tissue and cell culture

Transcription

1 mirna KIT Cat. No. EM12 Version: Kit for mirna isolation from animal tissue and cell culture EXTRACTME is a registered trademark of BLIRT S.A.

2 mirna KIT 2

3 Cat. No. EM12 I. INTENDED USE The EXTRACTME mirna KIT is designed for a rapid and efficient purification of mirna with possibility of simultaneous purification of large RNA and DNA. High quality mirna may be purified from 1-10 mg of tissue (fresh or frozen) and cultured cells. The isolation protocol, buffers formulations and columns were optimized for high isolation efficiency and purity of mirna. The product is intended for research use only. II. COMPONENTS OF THE KIT AND STORAGE CONDITIONS NUMBER OF ISOLATIONS 10 ISOLATIONS 50 ISOLATIONS 250 ISOLATIONS Storage conditions 1 Catalogue number EM EM EM mirlys Buffer * (mirna Lysis Buffer) 4 ml 20 ml 100 ml RT in dark mirw Buffer ** (Wash Buffer) mireb (Elution Buffer) 15 ml 2 x 37 ml 3 x 123 ml RT 3 ml 15 ml 5 x 15 ml RT DNA Purification Columns 10 pcs 50 pcs 5 x 50 pcs RT Large RNA Purification Columns 10 pcs 50 pcs 5 x 50 pcs RT mirna Purification Columns 10 pcs 50 pcs 5 x 50 pcs RT 1 RT room temperature (+15 C to +25 C) * For best efficiency during lysis of difficult material and for protection against RNases it is recommended to add 100% β-mercaptoethanol to mirlys Buffer, to a final concentration of 1%. The combined mirlys Buffer and β-mercaptoethanol will remain stable at 2 8 C for a period of four weeks. Therefore, while isolating in parts, transfer the amount of mirlys Buffer needed for one experiment to a separate RNase-free bottle/tube and add β-mercaptoethanol. Marking the bottle with added β-mercaptoethanol is recommended. ** Prior to the first use add an aropriate amount of % ethanol to mirw Buffer; for details, see the instructions on the bottle label as well as in the table below. Marking the bottle with added alcohol is recommended. Volume of mirw Buffer is calculated for isolation of DNA, RNA and mirna. If not all nucleic acids are isolated there is not need to use all bottles with mirw Buffer. 3

4 mirna KIT NUMBER OF ISOLATIONS 10 ISOLATIONS 50 ISOLATIONS 250 ISOLATIONS Catalogue number EM EM EM mirw Buffer * 15 ml 2 x 37 ml 3 x 123 ml % ethanol 35 ml 2 x 87 ml 3 x 287 ml Total volume 50 ml 2 x 124 ml 3 x 410 ml * While isolating mirna without DNA ang large RNA, diluted mirw buffer might be prepared in a smaller volume than given in the table. mirw Buffer should be diluted as follows: 1 volume of mirw Buffer to 2.33 volumes of ethanol. E. g. for 10 isolations without DNA and large RNA purification, use 4.5 ml mirw Buffer concentrate and 10.5 ml % ethanol. In order to avoid evaporation, ensure that the buffer bottles are tightly closed before storing. Protect mirlys Buffer from the sunlight! Expiry date Under proper storage conditions the kit will remain stable for at least 12 months from opening or until the expiry date. 4

5 Cat. No. EM12 III. ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED % ethanol PFA ml RNase-free microcentrifuge tubes automatic pipettes and pipette tips (RNase-free) disposable gloves microcentrifuge with rotor for ml ( x g) vortex mixer Might be necessary: 100% ß-mercaptoethanol scissors, scalpel bead-beating tubes with ceramic filling (cat. no. HPLM100, HPLM100a) tissue homogenizer for 2 ml tubes mechanical homogenizer with knives thermomixer, shaking orbit of 2 mm minimum ml smooth-stroke mortar with fitted piston liquid nitrogen or dry ice vortex mixer with a 2 ml tube adaptor centrifuge with a rotor for ml tubes (physiological fuids, cell cultures) 3% hydrogen peroxide or < 0.5% sodium hypochlorite IV. PRINCIPLE The EXTRACTME mirna KIT utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids at high concentration of chaotropic salts. During the first isolation step, the tissue is homogenized in order to disintegrate intercellular bonds (epithelial tissue) and fragmentize high-molecular proteins (muscle or connective tissue). A homogenate is lysed with guanidine thiocyanate and detergents. RNases are inactivated by guanidine thiocyanate and β-mercaptoethanol (optional). In next step DNA is separated by binding to the first minicolumn. Next large RNA is bound to a Large RNA Purifcation Column membrane by selective conditions in mixture after addition of ethanol. mirna is bound in next step to a mirna Purifcation Column. The three-step washing stage effectively removes impurities and enzyme inhibitors. Purified mirna is eluted using a low ionic strength buffer or RNase-free water (ph ) and can be used directly in all downstream alications such as RT-PCR, Northern blotting, RT-qPCR and so forth or stored until ready to use. 5

6 mirna KIT V. QUALITY CONTROL The quality of each production batch (LOT) of the EXTRACTME mirna KIT is tested using standard QC procedures. Purifed RNA concentration and quality are evaluated by gel electrophoresis and spectrophotometer. VI. PRODUCT SPECIFICATIONS SAMPLE MATERIAL fresh or frozen tissue (stored at -80 C): 1 10 mg tissue preserved in RNase inactivating buffers: 1 10 mg cell culture: cells BINDING CAPACITY ~ 90 μg mirna TIME REQUIRED minutes (lysis and homogenization time not included) minutes for homogenization in liquid nitrogen minutes for mechanical homogenization (ceramic beads) RNA PURITY A 260 /A 280 ratio =

7 Cat. No. EM12 VII. SAFETY PRECAUTIONS Tissue should be considered as a biohazardous material and treated as such on account of its potential pathogen content or health and life-threatening substances. While working with tissue and cell cultures it is essential to follow all safety requirements regarding work with biohazard material. It is recommended to carrying out the entire isolation procedure in the Class II Biological Safety Cabinet or at a laboratory burner as well as wearing disposable gloves and a suitable lab coat. It is recommended to use sterile RNase-free pipette tips. Avoid RNA transfer between minicolumns. Guanidine salts' residues may form highly reactive compounds when combined with oxidation components. In case of spillage, clean the surface with a detergent-water solution. In case of blood spillage, clean the surface first with detergent-water solution and next with 1% sodium hypochlorite. VIII. RECOMMENDATIONS AND IMPORTANT NOTES Quantity of starting material When isolating from greater than recommended amount of starting material (>10 mg, >10 6 cells), divide the material into several isolations so that each 10 mg (or 10 6 cells) portion is isolated with a separate buffer and minicolumn set. Exceeded quantity may clog a purification column and/or lower the purity of isolated mirna. Sampling and storing the material for RNA isolation Proper sampling and storing of biological material, prior to RNA isolation is crucial to obtain a high purity RNA. After sampling, the material should be preserved by deep freezing (at -80 C or in liquid nitrogen) or stored at -20 C in RNase inactivating buffers (e.g. RNAlater, Ambion). Most tissues should obligatory be preserved within 30 minutes of sampling. Tissues rich in RNases (pancreas, liver) require an immediate preservation. While isolating from cell cultures, best results are achieved with the use of fresh material. If storage is unavoidable, discard the supernatant after centrifugation and freeze the cell pellet at -80 C or in liquid nitrogen. 7

8 mirna KIT RNase elimination RNases are very active enzymes which do not require any cofactors and are resistant to 15 minutes autoclaving at 121 C. In order to avoid enzyme's degrading effect on RNA, it is essential to follow the recommendations below: a. Use disposable gloves at all times when working with RNA. Do not come in contact with any items that are not specifically designed to work with RNA. b. If possible, keep the samples at 2 8 C at all stages of the procedure, including centrifugation. Use decontaminated freezing racks instead of ice in order to avoid RNase contamination. Keeping large RNA and mirna after elution in the freezing racks is recommended. c. Plastic disposables (tips, tubes) should be RNase-free or autoclaved at 134 C for minutes. d. Reuseable plastic, glass and porcelain should be soaked overnight in 0.1 NaOH / 0.1% DEPC water (or RNase-free water) and then washed with 0.1% DEPC water (or RNase-free water). When alicable, glass and porcelain (mortars) should be parched at C for 2 4 h and cooled to room temperature. e. Wipe surfaces, pipettes, centrifuge (rotor should be wiped separately) and tube racks with 3% hydrogen peroxide or < 0.5% sodium hypochlorite (or any commercially available RNase inactivating fluid). Prior to decontamination, test the decontaminant on a small area of the material for possible undesired reactions. mirna elution The optimal volume of mireb (mirna Elution Buffer) used should be chosen accordingly to the amount of the sample material and the final nucleic acids concentration expected. Use of μl mireb is recommended. If a high nucleic acids concentration is desired, the elution volume may be reduced to 20 μl. It should be noted that this may reduce the efficiency of the nucleic acids recovery. It is essential to aly Elution Buffer precisely to the centre of the membrane. mireb does not contain EDTA, which may interfere with some enzymatic reactions. 8

9 Cat. No. EM12 RNA storage and stability For a long-term storage keep RNA at -80 C or in liquid nitrogen. DNA contamination All biological material used for RNA isolation contains DNA. There is no RNA isolation method that may guarantee a complete DNA removal unless RNA sample is treated with DNase after isolation. Even a slight DNA contamination (several gdna copies per reaction) may give an additional signal in a quantitative PCR analysis after reverse transcription. In order to avoid this, we recommend treating the purifed mirna sample with an aropriate enzyme. We also recommend designing primers which are insensitive to DNA contaminations (primers in adjoining exons or with intron >1.5 kbp) for the purposes of qpcr analysis. Foam formation in mirlys Buffer Due to detergent content of lysis buffer, it may create a foam after homogenization, vortexing or intensive pipetting. In order to avoid this, centrifuge at x g for 60 s. 9

10 mirna KIT IX. SAMPLE PREPARATION A. FRESH OR FROZEN SOLID TISSUE Quantity: 1-10 mg Sample material: animal or human tissues. General procedure, alies to all methods of homogenization Divide tissue into small fragments with tweezers and scissors or scalpel. Follow one of homogenization methods described below or go to step 1 of the Isolation Protocol (section XI). Liquid nitrogen, dry ice (LN 2, CO 2 ) 1. Put tissue frozen in LN 2 or CO 2 in a previously chilled, sterile mortar. Using a chilled piston, carefully, but firmly crush the tissue into smaller pieces and then, into a pulp. 2. Transfer the powder thus obtained into a 2 ml tube containing 400 μl mirlys Buffer and go to step 2 of the Isolation Protocol (section XI). ccafter pulping, a thin, sticky layer may be formed, rather than a powder. If this occurs, add 400 μl mirlys Buffer to a mortar and reconstitute the tissue by pipetting and then transfer the lysate into a sterile RNase-free 2 ml tube. Remember to retrieve a tissue remains from the piston as well. Homogenization using a mechanical homogenizer equied with knives 1. Place the tissue in a 2 ml tube, add 100 μl mirlys Buffer and carefully homogenize with a sterile homogenizer tip. 2. After homogenization, retrieve the tissue remains from the knife tip by washing it with 300 μl mirlys Buffer. Combine the fractions obtainded this way and transfer the entire volume to a new 2 ml tube. 3. Continue the isolation from step 2 of Isolation Protocol (section XI). Homogenization using bead-beating tubes 1. Add 100 μl mirlys Buffer to a 2 ml ceramic bead-beating tube and suspend the sliced tissue in the buffer. 2. Place the tube in the tissue homogenizer and homogenize at x g for 30 s. If necessary, repeat the procedure. ccif evaluation of the degree of tissue fragmentation is compromised by the foam formation, centrifuge the tube at x g for 60 s. ccif the tissue homogenizer is not available, the tissue may be homogenized by vortexing with the use of an aropriate 2 ml tube adaptor for at least 5 min at maximum speed. 10

11 Cat. No. EM12 3. Add 300 μl mirlys Buffer and mix by pipetting. 4. Continue the isolation from step 2 of Isolation Protocol (section XI). B. CELL CULTURES Quantity: cells Sample material: cell suspension or adherent cells, fresh or frozen. 1. Thaw frozen cells at 37 C. Centrifuge the cells suspended in a growth medium or PBS buffer in a 15 ml falcon tube or a ml Eendorf tube at 400 x g. If a compact cell pellet is not formed, wash the cells twice with 1 ml cold PBS buffer. 2. Add 400 μl mirlys Buffer. Mix thoroughly by vortexing for 30 s and subsequent pipetting. 3. Continue the isolation from step 2 of the Isolation Protocol (section XI). X. PRIOR TO ISOLATION 1. Mix well each buffer sulied with the kit. Do not mix mirlys Buffer vigorously. 2. Ensure that ethanol has been added to mirw Buffer. If not, add an aropriate amount of % ethanol (volumes can be found on the bottle labels or in the table given in section II). 3. Examine mirlys and mirw Buffers. If a sediment occurred in any of them, incubate it at 50 C (mirlys) or at 37 C (mirw) mixing occasionally until the sediment has dissolved. Cool to room temperature. OPTIONAL: 1. Prior to isolation add 100% ß-mercaptoethanol to mirlys Buffer to a final concentration of 1%. mirlys Buffer after β-mercaptoethanol was added is stable at 2 8 C for 4 weeks. Therefore, while isolating in parts, transfer an aropriate for one isolation amount of mirlys Buffer to a separate RNase-free bottle/tube and add β-mercaptoethanol. 2. Prepare freezing rack to store eluted RNA. 11

12 mirna KIT XI. ISOLATION PROTOCOL 1. Place a fragmented biological material in a 2 ml tube. Add 400 μl mirlys Buffer and vortex for 60 s. 2. Centrifuge for 120 s at x g. 3. Transfer the supernatant into DNA Purification Column placed in a collection tube. Centrifuge for 120 s at x g. Keep DNA Purification Column for further DNA purification. c c If not all the supernatant passes through the membrane, repeat the centrifugation for 120 s at x g. If the problem persist, it means that the material was insufficiently homogenized or the digestion time was too short or too much sample material was used for the isolation. 4. Transfer the filtrate into a sterile 1.5 ml Eendorf tube. 5. Add 0.5 volume of % ethanol. Mix by pipetting or vortexing for 5 s. c c For example to the 400 µl of the filtrate 200 µl ethanol should be added. 6. Transfer the mixture into Large RNA Purification Column placed in a collection tube. Centrifuge for 120 s at x g. Keep the filtrate. Keep Large RNA Purification Column for further large RNA purification. c c Minicolumn with large RNA can be stored no longer than 15 min at 4 8 C. 7. Transfer the filtrate into a sterile ml Eendorf tube. 8. Add 1 volume of % ethanol. Mix by pipetting or vortexing for 5 s. cc For example to the 600 µl of the filtrate 600 µl ethanol should be added. 9. Transfer 650 µl of the obtained mixture into mirna Purification Column placed in a collection tube. Centrifuge for 120 s at x g. Discard the filtrate and reuse the column, together with a collection tube. 12

13 Cat. No. EM Transfer the remaining mixture into the same mirna Purification Column and centrifuge for 120 s at x g. Discard the filtrate. 11. Prepare the minicolumns with bound DNA, large RNA and mirna. 12. Add 500 μl mirw Buffer to each prepared minicolumn and centrifuge for 60 s at x g. Discard the filtrate and reuse the collection tube. 13. Again add 500 μl mirw Buffer to each minicolumn and centrifuge for 60 s at x g. Discard the filtrate and reuse the collection tube. 14. Again add 500 μl mirw Buffer to each minicolumn and centrifuge for 60 s at x g. Discard the filtrate and reuse the collection tube. 15. Centrifuge for 3 min at x g. 16. Discard the collection tube and the filtrate and carefully transfer the minicolumns to a sterile 1.5 ml Eendorf tubes. 17. Add μl elution buffer mireb, precisely onto the centre of each purification minicolumn membrane. cc Other buffer volumes may be used. For instructions, see to section VIII. Recommendations and important notes. 18. Incubate the minicolumns at room temperature for 120 s. 19. Centrifuge for 120 s at x g. 20. Remove the minicolumns and place the tube with the eluted RNA in a freezing rack. The isolated RNA and DNA are ready for use in downstream alications or for storage at -80 C. 13

14 mirna KIT XII. TROUBLESHOOTING Problem Possible cause Solution Column becomes clogged during purification. Low mirna yield. Low purified nucleic acids concentration. Isolated DNA is of low purity Inaropriate tissue homogenization. The purification column is overloaded. Tissue and cell debris were transferred into the column. Tissue was incorrectly stored or preserved: RNA degradation. Too little sample material was used. Insufficient fragmentation of the sample material. The purification column has become clogged. RNases are present. Too much of elution buffer was used. RNA is still bound to the column membrane. One of the washing steps was omitted. Purified DNA contains residual alcohol. Select the aropriate homogenization conditions (see section IXA). Do not exceed the recommended tissue amount or number of cells taken for nucleic acids purification. Pipette the supernatant carefully, without disturbing the tissue or cell pellet. Store tissue at -80 C no longer than a year. If a tissue storage buffer was used, ensure if it was of a good quality and that the storage conditions were adequate. Take more sample material. A proper amount of the material is dependent on the kind of a cell line/tissue examined and needs to be optimized individually. Ensure proper tissue homogenization in mirlys Buffer. A tissue must be first fragmented into smallest possible pieces and homogenized by an aropriate method. See Column becomes clogged during purification. See RNase elimination in section VIII. Recommendations and Important Notes. Decrease mireb volume to μl. For a sample concentration it is possible to reload the eluate onto the column and centrifuge again. Repeat the RNA elution. Repeat the isolation, performing all three washing steps. Repeat the isolation, paying a particular attention to whether any residual mirw Buffer is left in column after final centrifugation step. 14

15 Cat. No. EM12 Purified large RNA is degraded. DNA contamination present in RNA sample. Old material was used. Material was repeatedly frozen/thawed. RNases are present. RNA degraded as a result of over-intensive homogenization. Too much sample material was used. Inaropriate homogenization. Performing an isolation from fresh tissues is recommended. Avoid subjecting the sample material to repeated freeze/thaw cycles. See RNase elimination in section VIII. Recommendations and Important Notes. The recommended homogenization conditions should be alied (see section IX). Decrease the amount of sample material. Optionally, the purified RNA sample can be treated with a DNase. The recommended homogenization conditions should be alied. XIII. SAFETY INFORMATION mirlys Buffer Danger H302, H331, H412 P261, P264, P301+P312 P330, P304+P340 P311, EUH032 EUH032 Contact with acids liberates very toxic gas. H302 Harmful if swallowed. H331 Toxic if inhaled. H412 Harmful to aquatic life with long lasting effects. P261 Avoid breathing dust/fumes/gas/mist/vapours/spray. P264 Wash hands thoroughly after handling. P301+P312 P330 IF SWALLOWED: Call a POISON CENTER/ doctor if you feel unwell. Rinse mouth. P304+P340 P311 IF INHALED: Remove person to fresh air and keep comfortable for breathing. Call a POISON CENTER/doctor. 15

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