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Total RNA isolation User manual

1. Proceed with step 5 of the NucleoSpin RNA II standard protocol section 5 1 MACHEREY NAGEL 03 2011 Rev 13 23 NucleoSpin RNA II 5 3 Support protocol NucleoSpin RNA II Total RNA preparation from up to 10 bacterial cells Additional reagent to be supplied by user Lysozyme Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 Homogenize sample Resuspend the bacterial cell pellet Gram negative strains in 100 uL TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 containing 1 mg mL lysozyme by vigorous vortexing Incubate at 37 C for 10 min For preparation of RNA from Gram positive bacteria resuspend cells in 100 uL TE containing 2 mg mL lysozyme It may be necessary to optimize incubation time and lysozyme concentration depending on the bacterial strain Note Due to the much higher concentration of genome equivalents in a nucleic acid preparation of bacteria compared with eukaryotic material it may be necessary to use a lower quantity of cells for the preparation Lyse cells Add 350 uL Buffer RA1 and 3 5 uL B mercaptoethanol to the suspension and vortex vigorously For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Filtrate lysate Reduce viscosity and turbidity of the so
2. 740606 740600 Suitable for 100 preps 50 mL 500 mL 1 set 107 mg 50 1000 DISTRIBUTION AND USE OF NUCLEOSPIN RNA DNA BUFFER SET AND NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS 44 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 6 3 Product use restriction warranty NucleoSpin RNA II kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for N VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for N VITRO diagnostic use Please pay attention t
3. Total RNA isolation User manual NucleoSpin RNA II NucleoSpin RNA L March 2011 Rev 13 MACHEREY NAGEL MN Total RNA isolation Protocol at a glance Rev 13 NucleoSpin RNA Il NucleoSpin RNA L 1 Homogenize sample 30 mg 100 mg 2 Lyse cells m 350 uL RA1 1 8 mL RA1 3 5 uL B mercaptoethanol 18 uL B mercaptoethanol Mix Mix 3 Filtrate lysate j 11 000 x g Y 4 500 x g 5 1 min j c 10 min 4 Adjust RNA 350 uL 70 ethanol 1 8 mL 70 ethanol binding i conditions Mix Mix 5 Bind RNA I Load sample B Load sample 5 11 000 x g 4 500 x g 30s A 3 min V V 6 Desalt silica membrane m 350 uL MDB 2 2 mL MDB 5 11 000 x g c 4 500 x g 1 min L 3 min 7 V 7 osi 95 uL DN 250 uL DN u ase L u ase E reaction mixture IY reaction mixture RT 15 min RT 15 min V 8 Wash and dry e silica ae dno P 1 wash 200 uLRA2 i i wash 2 6 mL RA2 2 wash 600 pL RA3 1 2 wash 2 6 mL RA3 D Ww 3 wash 250 uL RA3 V 3 wash 2 6 mLRA3 P Re 11 000 x g 1 and 2nd 4 500xg Wanda CD 30 s c2 8 min iad 11 000 x g gi 4 500xg c5 2 min 5 min 9 Elute highly 500 uL RNase pure RNA e SE O free H O ree H 3 3 RT 2 min 11 000 x g 1 min 4 500 x g 3 min MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net co
4. free Collection Tube 1 5 mL supplied 11 000 x g 2 min If for any reason the liquid level in the Collection Tube has c5 reached the NucleoSpin RNA II Column after centrifugation discard flow through and centrifuge again MACHEREY NAGEL 03 2011 Rev 13 21 NucleoSpin RNA II Elute RNA Elute the RNA in 60 uL RNase free H O supplied and centrifuge at 11 000 x g for 1 min If higher RNA concentrations are desired elution can be done with 40 uL Overall yield however will decrease when using smaller volumes For further alternative elution procedures see section 2 4 60 pL RNase free H O 11 000 x g 1 min 22 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA II 5 2 Support protocol NucleoSpin RNA II Total RNA preparation from biological fluids e g serum culture medium Before starting the preparation 1 Check if Wash Buffer RA3 and rDNase were prepared according to section 3 Homogenize sample Not necessary 2 Lysesample Add 350 uL Buffer RA1 and 3 5 uL B mercaptoethanol to 100 uL of sample and vortex vigorously For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 3 Filtrate lysate Not necessary 4 Adjust RNA binding conditions Add 350 uL of ethanol 70 to the lysate and mix by vortexing
5. Load lysate 11 000 x g 30s 350 pL MDB 11 000 x g 1 min MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA II Digest DNA Prepare DNase reaction mixture in a sterile 1 5 mL 95 uL microcentrifuge tube not provided For each isolation rDNase add 10 uL reconstituted rDNase also see section 3 to reaction 90 uL Reaction Buffer for rDNase Mix by flicking the mixture tube RT Apply 95 uL DNase reaction mixture directly onto the 15 min center of the silica membrane of the column Incubate at room temperature for 15 min Wash and dry silica membrane 1 wash 200 uL RA2 Add 200 uL BufferRA2 to the NucleoSpin RNA II 11 000 x g Column Centrifuge for 30 s at 11 000 x g Place the 30s column into a new Collection Tube 2 mL Buffer RA2 will inactivate the rDNase F Add 600 pL Buffer RA3 to the NucleoSpin RNA II C gt Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the Collection 600 uL RA3 Tube 11 000 x g Note Make sure that residual buffer from the previous steps 30s is washed away with Buffer RAS especially if the lysate has been in contact with the inner rim of the column during loading of the lysate onto the column For efficient washing of the inner rim flush it with Buffer RAS Add 250 uL Buffer RA3 to the NucleoSpin RNAI Column Centrifuge for 2 min at 11 000 x g to dry the 250 pL RA3 membrane completely Place the column into a nuclease
6. Adjust RNA binding conditions Discard the NucleoSpin Filter and add 350 pL ethanol 70 96 to the homogenized lysate and mix by pipetting up and down 5 times Alternatively transfer flow through into a new 1 5 mL microcentrifuge tube not provided add 350 uL ethanol 70 96 and mix by vortexing 2 x 5 s After addition of ethanol a stringy precipitate may become visible which will not affect the RNA isolation Be sure to disaggregate any precipitate by mixing and load all of the precipitate on the column as described in step 5 Do not centrifuge the ethanolic lysate before loading it onto the column in order to avoid pelleting the precipitate Bind RNA For each preparation take one NucleoSpin RNA II Column light blue ring placed in a Collection Tube Pipette lysate up and down 2 3 times and load the lysate to the column Centrifuge for 30 s at 11 000 x g Place the column in a new Collection Tube 2 mL Maximal loading capacity of NucleoSpin RNA II Columns is 750 uL Repeat the procedure if larger volumes are to be processed Desalt silica membrane Add 350 uL MDB Membrane Desalting Buffer and centrifuge at 11 000 x g for 1 min to dry the membrane Salt removal will make the following rDNase digest much more effective If the column outlet has come into contact with the flow through for any reason discard the flow through and centrifuge again for 30 s at 11 000 x g 350 uL 70 ethanol Mix
7. Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 Homogenize sample Disrupt up to 100 mg of tissue for sample amounts see section 2 2 for homogenization methods see section 2 3 c Disrupt sample Up to 4 x 107 eukaryotic cultured cells are collected by P centrifugation and lysed by addition of Buffer RA1 directly To choose an appropriate amount of starting material see section 2 2 2 Lysecells Add 1 8 mL Buffer RA1 and 18 uL B mercaptoethanol B ME to the disrupted material in a 15 mL centrifuge tube not supplied and vortex vigorously use 3 6 mL 1 8 mL RA1 Buffer RA1 and 36 uL B mercaptoethanol for large sample amounts see section 2 2 18 uL B ME Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 3 Filtrate lysate Apply the lysate to a NucleoSpin Filter L placed in a Collection Tube and centrifuge sample for 10 min at 4 500 x g This step will homogenize the sample by removal of residual insoluble material and simultaneous reduction of lysate viscosity In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to anew 15 mL centrifuge tube not supplied lt C ji gt 4 500 x g If working with small amounts of cultured cells e g lt 1 x 10 e gt 10 min HeL
8. Concentrate 25 mL Membrane Desalting Buffer MDB 50 mL Reaction Buffer for rDNase 7 mL rDNase RNase free lyophilized 1 vial size D RNase free H O 15 mL NucleoSpin Filters L 20 plus Collection Tubes NucleoSpin RNA L Columns 20 plus Collection Tubes Collection Tubes 15 mL 20 User manual 1 For preparation of working solutions and storage conditions see section 3 6 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol to prepare Wash Buffer RA3 70 ethanol to adjust RNA binding conditions Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane as supplement for Lysis Buffer RA1 Consumables 1 5 mL microcentrifuge tubes NucleoSpin RNAI or 15 mLtubes NucleoSpin RNAL Sterile RNase free tips Equipment Manual pipettors NucleoSpin RNA II centrifuge for microcentrifuge tubes NucleoSpin RNA L centrifuge for 15 mL tubes with a swing out rotor and appropriate buckets capable of reaching 4 000 4 500 x g Equipment for sample disruption and homogenization see section 2 3 Personal protection equipment e g lab coat gloves goggles 1 3 About this user manual It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoSpin RNA II or NucleoSpin RNA L kit is used for the fir
9. NucleoSpin RNA II and NucleoSpin RNA L rDNase digestion in solution NN OO 13 14 15 17 19 19 23 24 26 28 29 33 34 36 38 39 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty 41 41 44 45 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 1 Components 1 1 Kit contents NucleoSpin RNA II 10 preps 20 preps 50 preps 250 preps REF 740955 10 740955 20 740955 50 740955 250 Lysis Buffer RA1 10 mL 10 mL 25 mL 125 mL Wash Buffer RA2 15 mL 15 mL 15 mL 80 mL Wash Burer HAS 5mL 5mL 12 5 mL 3x25mL Concentrate Membrane Desalting Buffer MDB 10 mL 10 mL 25 mL 125 mL Reaction Buffer Smb 3 mL 7 mL 30 mL for rDNase rDNase RNase free 1 vial 1 vial 1 vial 5 vials lyophilized size C size C size D size D RNase free H O 5 mL 5 mL 15 mL 65 mL ee NucleoSpin Filters 10 20 50 250 violet rings NucleoSpin RNA Il Columns light 10 20 50 250 blue rings plus Collection Tubes Collection Tubes 30 60 150 750 2 mL Collection Tubes 1 5 mL 10 20 50 250 User manual 1 1 1 1 For preparation of working solutions and storage conditions see section 3 MACHEREY NAGEL 03 2011 Rev 13 5 Total RNA isolation 1 1 Kit contents continued NucleoSpin RNA L 20 preps REF 740962 20 Lysis Buffer RA1 80 mL Was Buffer RA2 60 mL Wash Buffer RA3
10. The RNA prepared from such high amounts is generally free of residual DNA although minute traces of DNA may remain in the preparation if large amounts of material rich in nucleic acids are used However if the isolated RNA will be used as template in a RT PCR reaction we recommend to use lower quantities of sample e g 1 x 10 cultured cells or 10 mg of tissue resulting in about 20 ug of RNA The kit can be used for preparing RNA from different amounts of sample material For optimal results the volume of Lysis Buffer RA1 protocol step 1 and of ethanol protocol step 3 should be adapted according to Table 2 10 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation Table 2 Lysis adaptation Sample Amount Volume of Lysis Buffer RA1 Ethanol protocol step 2 protocol step 4 Cultured animal lt 5x 109 350 uL 350 uL or human cells e g HeLa cells Human or 20 mg 350 uL 350 uL animal tissue 20 mg 30 mg 600 uL 600 uL Tissue stored 20 mg 350 uL 350 uL in RNA ater 20 mg 30 mg 600 uL 600 uL Samples known 5x107 600 uL 600 uL to be hard to lyse An additional loading step is required if 600 uL Buffer RA1 and ethanol is used load the sample onto the column in two successive centrifugation steps Depending on sample type the average yield is around 5 70 pg total RNA see Table 3 The Asgo Azgo ratio generally exceeds 1 9 indicating purity of the RNA Table 3 Overview on avera
11. an enzymatic digestion A or mechanical homogenization B depending on laboratory equipment and personal preference Homogenization by enzymatic digest is only recommended for fresh harvested cells homogenization by mechanical disruption may also be performed with yeast cell pellets stored at 70 C for several months Note Due to the much higher concentration of genome equivalents in a nucleic acid preparation of yeasts compared with cultured cells or tissue material it may be necessary to use a lower quantity of cells for the preparation A Homogenization by enzymatic digest Harvest 2 5 mL of YPD culture 5 000 x g 10 min Resuspend pellet in an appropriate amount of fresh prepared sorbitol lyticase buffer 50 100 U lyticase or zymolase in 1 M sorbitol 100 mM EDTA and incubate at 30 C for 30 min Pellet the resulting spheroplasts by centrifugation 1 000 x g 10 min Carefully discard supernatant It may be necessary to optimize incubation time and lyticase zymolase concentration depending on the yeast strain Continue with step 2 OR B Homogenization by mechanical disruption Harvest 2 5 mL of YPD culture 5 000 x g 10 min and wash with ice cold water Resuspend the cell pellet in a mixture of 350 uL Buffer RA1 and 3 5 uL B mercaptoethanol Add glass beads e g 300mg glass beads 425 600 um Sigma Aldrich 68772 26 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA II Shake samples in a swing
12. are given for homogenization of yeast cells Users may choose between an enzymatic digestion A or mechanical homogenization B depending on laboratory equipment and personal preference Homogenization by enzymatic digest is only recommended for fresh harvested cells homogenization by mechanical disruption may also be performed with yeast cell pellets stored at 70 C for several months Note Due to the much higher concentration of genome equivalents in a nucleic acid preparation of yeasts compared with cultured cells or tissue material it may be necessary to use a lower quantity of cells for the preparation A Homogenization by enzymatic digest Harvest an appropriate amount of cells from YPD culture 5 000 x g 10 min Resuspend pellet in an appropriate amount of fresh prepared sorbitol lyticase buffer 50 100 U Iyticase or zymolase in 1 M sorbitol 100 mM EDTA and incubate at 30 C for 30 min Pellet the resulting spheroplasts by centrifugation 1 000 x g 10 min Carefully discard suprnatant It may be necessary to optimize incubation time and lyticase zymolase concentration depending on the yeast strain Continue with step 2 OR B Homogenization by mechanical disruption Harvest an appropriate amount of cells from YPD culture 5 000 x g 10 min and wash with ice cold water Resuspend the cell pellet in a mixture of 3 6 mL Buffer RA1 and 36 uL 8 mercaptoethanol Add glass beads e g 300 mg glass beads 425 600 um Si
13. contaminations of RNases often found on general lab ware fingerprints and dust To ensure RNA stability keep RNA frozen at 20 C for short term or 70 C for long term storage Simultaneous isolation of RNA Protein and DNA NucleoSpin RNA DNA Buffer Set NucleoSpin TriPrep The NucleoSpin RNA DNA Buffer Set see ordering information is a support set for RNA and DNA isolation in conjunction with NucleoSpin RNA II NucleoSpin RNA XS NucleoSpin RNA Plant or NucleoSpin RNA Protein This patented technology enables successive elution of DNA and RNA from one NucleoSpin Column with low salt buffer and water respectively DNA and RNA are immediately ready for downstream applications The combination of the NucleoSpin RNA DNA Buffer Set with NucleoSpin RNA Protein allows parallel isolation of RNA DNA and protein from one undivided sample The NucleoSpin TriPrep kit features the purification of RNA DNA and protein from single undivided samples DISTRIBUTION AND USE OF NUCLEOSPIN RNA DNA BUFFER SET AND NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS 8 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 2 2 Kit specifications NucleoSpin RNA kits are recommended for the isolation of total RNA from cultured cells and tissue Support protocols for the isolation of total RNA from cell free biological fluids bacteria and yeasts using the NucleoSpin RNA II kit are included The NucleoS
14. mill at 30 Hz for 15 min Continue with step 3 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Lyse cells Add 350 uL Buffer RA1 and 3 5 uL B mercaptoethanol and vortex vigorously to lyse spheroplasts For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Filtrate lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filters violet rings Place NucleoSpin Filters in Collection Tubes 2 mL apply mixture and centrifuge for 1 min at 11 000 x g In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 1 5 mL microcentrifuge tube not supplied Alternatively the lysate may be passed z 5 times through a 0 9 mm needle 20 gauge fitted to a syringe Adjust RNA binding conditions Add 350 uL of ethanol 70 to the lysate and mix by vortexing Proceed with step 5 of the NucleoSpin RNA II standard protocol section 5 1 MACHEREY NAGEL 03 2011 Rev 13 27 NucleoSpin RNA L 5 5 Support protocol NucleoSpin RNA II Total RNA preparation from paraffin embedded tissue Additional reagent to be supplied by user Xylene Before start
15. probability of DNA detection with PCR increases with 1 the number of DNA copies per preparation single copy target plastidial mitochondrial target plasmid transfected into cells 2 decreasing PCR amplicon size MACHEREY NAGEL 03 2011 Rev 13 9 Total RNA isolation Table 1 Kit specifications at a glance Parameter NucleoSpin RNA II NucleoSpin RNA L Sample material lt 5 x 10 cultured cells lt 5 x 107 cultured cells 10 bacterial cells lt 101 bacterial cells 10 yeast cells 3 x 108 yeast cells lt 30 mg tissue 200 mg tissue Format Mini spin column Midi spin column Fragment size gt 200 b gt 200 b Typical yield 14 ug from 109 HeLa cells 180 ug from 10 HeLa cells 70 ug from 10 bacterial cells 620 ug from 4 x 107 HeLa cells Acsi Asse 1 9 2 1 1 9 2 1 Typical RIN RNA gt 9 gt 9 integrity number Elution volume 40 120 uL 500 uL Binding capacity 200 ug 700 ug Preparation time 30 min 6 preps 80 min 4 preps NucleoSpin RNA II The standard protocol section 5 1 allows the purification of up to 70 ug of total RNA per NucleoSpin RNA II Column from up to 5 x 10 cultured cells or 30 mg of tissue also see Table 1 The isolated RNA can be used as template in a RT PCR reaction Generally 1 10 96 of the eluate of total RNA prepared from 1 x 108 cells or 10 mg of tissue is sufficient as template for RT PCR If possible intron spanning primers should be used for RT PCR
16. reaction mixture RT 15 min 2 6 mL RA2 4 500x g 3 min 2 6 mL RA3 4 500x g 3 min 2 6 mL RA3 4 500 x g 5 min MACHEREY NAGEL 03 2011 Rev 13 31 NucleoSpin RNA L Elute RNA Pipette 500 uL RNase free H O supplied directly onto the center of the silica membrane Incubate at room temperature for 2 min and centrifuge for 3 min at 4 500 x g Reduction of elution volume will generally not result in an increased concentration of eluted nucleic acid with the NucleoSpin RNA L kit see section 2 4 for alternative elution procedures 6 500 uL RNase free H O RT 2 min 4 500 x g 3 min 32 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA L 5 7 Support protocol NucleoSpin RNA L Total RNA preparation from up to 5 x 10 bacterial cells Additional reagent to be supplied by user Lysozyme Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 Homogenize sample Resuspend the bacterial cell pellet Gram negative strains in 200 uL TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 containing 1 mg mL lysozyme by vigorous vortexing Incubate at 37 C for 10 min For preparation of RNA from Gram positive bacteria resuspend cells in 200 uL TE containing 2 mg mL lysozyme It may be necessary to optimize incubation time and lysozyme concentration depending on the bacterial strain 2 Lysecells Add 1 8 mL Buffer RA1
17. thiocyanat 1096 Ethanol 1096 Hazard labeling not neccessary if quantity per bottle below 125g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 8 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet MACHEREY NAGEL 03 2011 Rev 13 17 NucleoSpin RNA II Risk phrases R S tze R10 R 20 21 22 R 42 43 Flammable Entztindlich Harmful by inhalation in contact with skin and if swallowed Gesundheitssch dlich beim Einatmen Verschlucken und Ber hrung mit der Haut May cause sensitisation by inhalation and skin contact Sensibilisierung durch Einatmen und Hautkontakt m glich Safety phrases S S tze S7 13 16 S22 S24 Keep container tightly closed Beh lter dicht geschlossen halten Keep away from food drink and animal feedstuffs Von Nahrungsmitteln Getr nken und Futtermitteln fernhalten Keep away from sources of ignition No Smoking Von Z ndquellen fernhalten Nicht rauchen Do not breathe dust Staub nicht einatmen Avoid contact with the skin Ber hrung mit der Haut vermeiden 18 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA II 5 Protocols 5 1 Total RNA purification from cultured cells and tissue with NucleoSpin RNA II Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 Homogenize sample Di
18. a cells step 3 may be substituted by vigorous mixing of the sample MACHEREY NAGEL 03 2011 Rev 13 29 NucleoSpin RNA L Adjust RNA binding conditions Discard the NueoSpin Filter L and add 1 8 mL ethanol 7096 to the lysate in the Collection Tube and mix by vortexing 2 x 5 s use 3 6 mL of 70 ethanol if working with large sample amounts see step 2 and section 2 2 After addition of ethanol a stringy precipitate may become visible which will not affect the further procedure Resuspend precipitates thoroughly before loading onto the NucleoSpin RNA L Column Bind RNA Load the lysate ethanol mixture maximal 3 8 mL onto a NucleoSpin RNA L Column Centrifuge for 3 min at 4 500 x g If working with large sample amounts apply the rest of the lysate ethanol mixture max 3 8 mL onto the column and centrifuge again If the lysate has not passed through the column centrifuge again at 4 500 x g for 10 min In case of column overloading incomplete flow through of the sample might be observed for example the membrane is still wet or some lysate has not passed through Remove the lysate which has not passed through the column and continue with the next protocol step Use less starting material and carefully remove insoluble material in step 3 next time Desalt silica membrane Add 2 2 mL MDB Membrane Desalting Buffer to the NucleoSpin RNA L Column Centrifuge for 3 min at 4 500 x g Discard flow thr
19. al DNA This DNA will not be detectable in most downstream applications Despite this there are still certain applications which require even lower contents of residual DNA However removal of DNA to a completely undetectable level is challenging and the efficiency of an on column DNA digestion is sometimes not sufficient for downstream applications requiring lowest residual content of DNA A typical example for such a demanding application is an RT PCR reaction in which the primer molecules do not differentiate between cDNA derived from RNA and contaminating genomic DNA Especially if high copy number targets are analyzed e g multi gene family mitochondrial plastidal or plasmid targets from transfections the target gene is of a very low expression level the amplicon is relatively small 200 bp DNA digestion in solution can efficiently destroy contaminating DNA However stringent RNase control and subsequent repurification of the RNA in order to remove buffer salts DNase and digested DNA are usually required The high quality recombinant RNase free DNase rDNase inthe NucleoSpin RNA kits facilitates such a digestion in solution in order to remove even traces of contaminating DNA A Digest DNA Reaction setup Add 6 uL Reaction Buffer for rDNase and 0 6 uL rDNase to 60 uL eluted RNA Alternatively premix 100 uL Reaction Buffer for rDNase and 10 uL rDNase and add 1 10 volume to one volume of RNA eluate G
20. and 18 uL B mercaptoethanol to the suspension and vortex vigorously For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 3 filtrate lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filters L Apply the lysate to a NucleoSpin Filter L placed in a Collection Tube and centrifuge for 10 min at 4 500 x g In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 15 mL centrifuge tube not supplied Proceed with step 4 of the NucleoSpin RNA L standard protocol section 5 6 MACHEREY NAGEL 03 2011 Rev 13 33 NucleoSpin RNA Il NucleoSpin RNA L 5 8 Support protocol NucleoSpin RNA L Total RNA preparation from up to 3 x 10 yeast cells Additional reagents and components to be supplied by user Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane Sorbitol and lyticase or zymolase for homogenization by enzymatic digestion or a swing mill and glass beads for homogenization by mechanical disruption Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 Homogenize sample Two alternative protocols
21. ction mixtures Before starting the preparation 1 Check if Wash Buffer RA3 was prepared according to section 3 Prepare sample Fill up RNA samples smaller than 100 uL with RNase free H O to 100 uL If different samples with varying volumes between 100 and 200 pL are purified RNA samples should be filled up with RNase free H O to a uniform volume e g 200 pL Prepare lysis binding buffer premix Prepare a Buffer RA1 ethanol premix with ratio 1 1 For each 100 uL RNA sample mix 300 pL Buffer RA1 and 300 uL ethanol 96 100 If multiple samples are processed the preparation of a master premix is recommended e g 2 mL RA1 2 mL 98 ethanol for approximately 6 prepara tions Filtrate lysate Not necessary Adjust RNA binding conditions To 100 uL of RNA sample add 600 uL 6 volumes of Buffer RA1 ethanol premix Mix sample with premix by vortexing If 200 uL of RNA samples are processed add 1200 uL of RA1 ethanol premix Maximal loading capacity of NucleoSpin RNA II Columns is 750 uL Repeat the procedure if larger volumes are to be processed After addition of ethanol a stringy precipitate may become visible which will not affect the RNA isolation Be sure to mix thoroughly and apply sample as homogenious solution onto the column 36 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA Il NucleoSpin RNA L Proceed with step 5 8 and 9 of the NucleoSpin RNA II standard prot
22. demarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 46 MACHEREY NAGEL 03 2011 Rev 13
23. ently swirl the tube in order to mix the solution Spin down gently approx 1 s at 1 000 x g to collect every droplet of the solution at the bottom of the tube B Incubate sample Incubate for 10 min at 37 C MACHEREY NAGEL 03 2011 Rev 13 39 Total RNA isolation C Repurify RNA Repurify RNA with a suitable RNA cleanup procedure for example by use of the NucleoSpin RNA Clean up NucleoSpin RNA Clean up XS kits see ordering information or by ethanol precipitation Ethanol precipitation exemplary Add 0 1 volume of 3 M sodium acetate pH 5 2 and 2 5 volumes of 96 100 ethanol to one volume of sample Mix thoroughly Incubate several minutes to several hours at 20 C or 4 C Note Choose long incubation times if the sample contains low RNA concentration Short incubation times are sufficient if the sample contains high RNA concentration Centrifuge for 10 min at maximum speed Wash RNA pellet with 7096 ethanol Dry RNA pellet and resuspend RNA in RNase free H O 40 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 6 Appendix 6 1 Troublesho oting Problem Possible cause and suggestions RNase contamination RNA is degraded no RNA obtained Create an RNase free working environment Wear gloves during all steps of the procedure Change gloves frequently Use of sterile disposable polypropylene tubes is recommended Keep tubes closed whenever possible during the pr
24. eparation Glassware should be oven baked for at least 2 hours at 250 C before use Reagents not applied or restored properly Reagents not properly restored Add the indicated volume of RNase free H O to rDNase vial and 96 ethanol to Buffer RA3 Concentrate and mix Reconstitute and store lyophilized rDNase according to instructions given in section 3 Sample and reagents have not been mixed completely Always vortex vigorously after each reagent has been added No ethanol has been added after lysis Binding of RNA to the silica membrane is only effective in the presence of ethanol Kit storage Poor RNA quality or yield Reconstitute and store lyophilized rDNase according to instructions given in section 3 Store other kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in order to prevent evaporation or contamination lonic strength and pH influence Azs absorption as well as ratio A ogo A280 For adsorption measurement use 5mM Tris pH 8 5 as diluent Please see also Manchester K L 1995 Value of A go go ratios for measurement of purity of nucleic acids Biotechniques 19 208 209 Wilfinger W W Mackey K and Chomczyski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity Biotechniques 22 474 481 MACHEREY NAGEL 03 2011 Rev 13 41 Total RNA isolation Poor RNA
25. free RNase free RNase free RNase free H O H O HO HO to each vial NucleoSpin RNA L 20 preps REF 740962 20 Wash Buffer RA3 Concentrate 25 mL Add 100 mL ethanol rDNase RNase free lyophilized 1 vial size D Add 540 uL RNase free H O 16 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 4 Safety instructions risk and safety phrases The following components of the NucleoSpin RNA II and NucleoSpin RNA L kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Hazard Risk Safety contents symbol phrases phrases Inhalt Gefahrstoff Gefahrstoffsymbol R S tze S S tze rDNase rDNase x May cause sensitization by R 42 43 S 22 24 RNase free lyophilized Xn inhalation and skin contact rDNase Sensibilisierung durch Einatmen lyophilisiert und Hautkontakt m glich RA1 Guanidinium x Harmful by inhalation in R 20 21 22 S 13 thiocyanate Xn contact with the skin and if swallowed Guanidinium Gesundheitssch dlich beim thiocyanat Einatmen Verschlucken und Ber hrung mit der Haut RA2 Guanidinium x Flammable Harmful by in R 10 S 7 18 16 thiocyanate Xn halation in contact with skin 20 21 22 and if swallowed Guanidinium Entz ndlich Gesundheitssch d thiocyanat lich beim Einatmen Verschlucken und Ber hrung mit der Haut MDB Guanidinium Flammable R 10 S 7 16 thiocyanate 1096 ethanol 1096 Guanidinium Entz ndlich
26. ge yields of total RNA isolation using NucleoSpin RNA II Sample Average yield 8 x 10 HeLa cells 1 5 ug 4 x 10 HeLa cells 4 ug 1 x 106 HeLa cells 14 ug 2 x 10 HeLa cells 21 ug 2 5 x 10 HeLa cells 25 ug 5 x 10 HeLa cells 50 ug The volume of Lysis Buffer RA1 included in the kit is not sufficient to perform all preparations with 600 uL If required additional Lysis Buffer RA1 can be ordered separately see ordering information MACHEREY NAGEL 03 2011 Rev 13 11 Total RNA isolation NucleoSpin RNA L The kit can be used for preparing RNA from different amounts of sample material For optimal results the volume of Lysis Buffer RA1 protocol step 1 and of ethanol protocol step 3 should be adapted according to table 4 Table 4 Lysis adaptation Sample Amount Volume of Lysis Buffer RA1 Ethanol protocol step 1 protocol step 4 Cultured animal cells 5x 10 2 x 107 1 8 mL 1 8 mL e g HeLa cells 2x 107 5 x 10 3 6 mL 3 6 mL Animal tissue 30 100 mg 1 8 mL 1 8 mL 100 200 mg 3 6 mL 3 6 mL Bacteria 1 x 107 5 x 10 1 8 mL 1 8 mL 2 x 102 1 x 1079 3 6 mL 3 6 mL Yeast 3 x 10 3 6 mL 3 6 mL An additional loading step is required if 3 6 mL Buffer RA1 and ethanol is used If you isolate RNA from a certain kind of tissue the first time with the NucleoSpin RNA L kit we recommend starting with no more than 100 mg of tissue Depending on the nature of the tissue up t
27. gma Aldrich 68772 34 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA Il NucleoSpin RNA L Shake samples in a swing mill at 30 Hz for 15 min Continue with step 3 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Lyse cells Add 3 6 mL Buffer RA1 and 36 uL B mercaptoethanol and vortex vigorously to lyse spheroplasts For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Filtrate lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filter L Place NucleoSpin Filter L placed in Collection Tubes and centrifuge for 10 min at 4 500 x g In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 15 mL centrifuge tube not supplied Adjust RNA binding conditions Discard the NucleoSpin Filter L and add 3 6 mL 7096 ethanol to the lysate in the Collection Tube and mix by vortexing Proceed with step 5 of the NucleoSpin RNA L standard protocol section 5 6 MACHEREY NAGEL 03 2011 Rev 13 35 NucleoSpin RNA Il NucleoSpin RNA L 5 9 Support protocol NucleoSpin RNA Il and NucleoSpin RNA L Clean up of RNA from rea
28. h touch the column outlet after the second Buffer RA3 wash Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic Buffer RA3 completely Suboptimal Checkif Buffer RA3 has been equilibrated to room temperature performance before use Washing at lower temperatures lowers efficiency of RNA in of salt removal by Buffer RAS downstream experiments Store isolated RNA properly Eluted RNA should always be kept on ice for optimal stability since trace contaminations of omnipresent RNases general lab ware fingerprints dust will degrade the isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C MACHEREY NAGEL 03 2011 Rev 13 43 Total RNA isolation 6 2 Ordering information Product REF Pack of NucleoSpin RNA II 740955 10 20 50 250 10 20 50 250 NucleoSpin RNA L 740962 20 20 NucleoSpin RNA Protein 740933 10 50 250 10 50 250 NucleoSpin TriPrep 740966 10 50 250 10 50 250 NucleoSpin FFPE RNA 740969 10 50 250 10 50 250 NucleoSpin RNA Clean up 740948 10 50 250 10 50 250 NucleoSpin RNA XS 740902 10 50 250 10 50 250 NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 NucleoSpin RNA Buffer Set Buffer RA1 Buffer RA1 rDNase Set TCEP NucleoSpin Filters Collection Tubes 2 mL Visit www mn net com for more detailed product information 740944 740961 740961 500 740963 740395 107
29. hich are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or MACHEREY NAGEL 03 2011 Rev 13 45 Total RNA isolation components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed
30. ing the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 A Put 10 mg of finely minced tissue into a 1 5 mL microcentrifuge tube not provided Add 300 uL xylene and incubate 5 min with constant mixing at room temperature B Centrifuge at maximum speed 13 000 rpm for 3 min to pellet the tissue Discard the xylene C Repeat the steps A and B twice for a total of three xylene washes D Add 300 pL of 96 ethanol to the tube and incubate 5 min with constant mixing at room temperature E Centrifuge at maximum speed 13 000 rpm for 3 min to pellet the tissue Discard the ethanol F Repeat steps D and E for a total of two ethanol washes Continue with step 1 of the NucleoSpin RNA II standard protocol section 5 1 Note For high performance isolation of RNA from formalin fixed paraffin embedded tissue the NucleoSpin FFPE RNA kit REF 740969 see ordering information is recommended Please also refer to Annunziata Gloghini Barbara Canal Ulf Klein Luigino Dal Maso Tiziana Perin Riccardo Dalla Favera and Antonino Carbone RT PCR Analysis of RNA Extracted from Bouin Fixed and Paraffin Embedded Lymphoid Tissues J Mol Diagn 2004 6 290 296 as one example for customer modification of the support protocol mentioned above 28 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA L 5 6 Total RNA purification from cultured cells and tissue with NucleoSpin RNA L
31. lution by filtration through NucleoSpin Filters violet rings Place NucleoSpin Filters in Collection Tubes 2 mL apply mixture and centrifuge for 1 min at 11 000 x g In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 1 5 mL microcentrifuge tube not supplied Alternatively the lysate may be passed gt 5 times through a 0 9 mm needle 20 gauge fitted to a syringe 24 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA II 4 Adjust RNA binding conditions Add 350 uL of ethanol 70 to the lysate and mix by vortexing Proceed with step 5 of the NucleoSpin RNA II standard protocol section 5 1 MACHEREY NAGEL 03 2011 Rev 13 25 NucleoSpin RNA II 5 4 Support protocol NucleoSpin RNA II Total RNA preparation from up to 5 x 10 yeast cells Additional reagents and components to be supplied by user Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane Sorbitol and lyticase or zymolase for homogenization by enzymatic digestion or a swing mill and glass beads for homogenization by mechanical disruption Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 Homogenize sample Two alternative protocols are given for homogenization of yeast cells Users may choose between
32. m Total RNA isolation Table of contents 1 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 1 3 About this user manual 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Handling preparation and storage of starting materials 2 4 Elution procedures 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 Protocols 5 1 Total RNA purification from cultured cells and tissue with NucleoSpin RNA II 5 2 Support protocol NucleoSpin RNA II Total RNA preparation from biological fluids e g serum culture medium 5 3 Support protocol NucleoSpin RNA Il Total RNA preparation from up to 10 bacterial cells 5 4 Support protocol NucleoSpin RNA II Total RNA preparation from up to 5 x 10 yeast cells 5 5 Support protocol NucleoSpin RNA Il Total RNA preparation from paraffin embedded tissue 5 6 Total RNA purification from cultured cells and tissue with NucleoSpin RNA L 5 7 Support protocol NucleoSpin RNA L Total RNA preparation from up to 5 x 10 bacterial cells 5 8 Support protocol NucleoSpin RNA L Total RNA preparation from up to 3 x 10 yeast cells 5 9 Support protocol NucleoSpin RNA II and NucleoSpin RNA L Clean up of RNA from reaction mixtures 5 10 Support protocol NucleoSpin RNA II and NucleoSpin RNA L Total RNA preparation from RNA ater treated samples 5 11 Support protocol
33. o 200 mg can be processed Do not use more than 200 mg of tissue to avoid clogging of the column Depending on sample type the average yield is around 70 400 pg total RNA see table 5 The Azgo Azgo ratio indicating purity of the RNA generally exceeds 1 9 Table 5 Overview on average yields of total RNA isolation using NucleoSpin RNA L Sample Average yield 1x 10 HeLa cells 20 ug 1 x 10 HeLa cells 160 ug 2 x 10 HeLa cells 330 ug 4 x 10 HeLa cells 620 ug 200 mg pig liver 450 ug 200 mg mouse liver 320 ug 12 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 2 3 Handling preparation and storage of starting materials RNA is not protected against digestion until the sample material is flash frozen or disrupted in the presence of RNase inhibiting or denaturing agents Therefore it is important that samples are flash frozen in liquid N immediately and stored at 70 C Or processed as soon as possible Samples can be stored in Lysis Buffer RA1 after disruption at 70 C for up to one year at 4 C for up to 24 hours or up to several hours at room temperature Frozen samples are stable up to 6 months Frozen samples in Buffer RA1 should be thawed slowly before starting with the isolation of total RNA Wear gloves at all times during the preparation Change gloves frequently Cultured animal cells are collected by centrifugation and directly lysed by adding Buffer RA1 according to step 2 of the
34. o the package of the product N VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR N VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL w
35. ocol section 5 1 or with step 5 8 and 9 of NucleoSpin RNA L standard protocol section 5 6 Steps 6 and 7 of the respective protocols may be omitted in this case As alternative products for RNA clean up NucleoSpin RNA Clean up and NucleoSpin RNA Clean up XS are recommended see ordeering information MACHEREY NAGEL 03 2011 Rev 13 37 NucleoSpin RNA Il NucleoSpin RNA L 5 10 Support protocol NucleoSpin RNA Il and NucleoSpin RNA L Total RNA preparation from RNA ater treated samples Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 Prepare sample Remove RNAlater solution Cut an appropriate amount of tissue 2 Lyse cells Add 350 uL NucleoSpin RNA 11 1 8 mL NucleoSpin RNA L Buffer RA1 and 3 5 uL NucleoSpin RNA 11 18 pL NucleoSpin RNA L B mercaptoethanol to the sample Disrupt the sample material by using for example rotor stator homogenizers for homogenization methods see section 2 4 Proceed with step 3 filtrate lysate of the NucleoSpin RNA II standard protocol section 5 1 or NucleoSpin RNA L standard protocol section 5 6 38 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 5 11 Support protocol NucleoSpin RNA Il and NucleoSpin RNA L rDNase digestion in solution The on column rDNase digestion in the standard protocol is already very efficient and thus resulting in minimal residu
36. or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio 9 mn net com Trademarks disclaimer RNAlater is a registered trademark of AMBION Inc NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands tra
37. ough Ifthe silica membrane is notcompletely dry after centrifugation centrifuge again at 4 500 x g for 10 min This step will create optimal reaction conditions for the rDNase 9 1 8 mL 70 ethanol Mix Load max 3 8 mL lysate 4 500 x g 3 min 2 2 mL MDB 4 500 x g 3 min 30 MACHEREY NAGEL 03 2011 Rev 13 NucleoSpin RNA L 7 Digest DNA Prepare DNase reaction mixture in a sterile microcen trifuge tube mix 235 uL Reaction Buffer for rDNase and 25 uL reconstituted rDNase see section 3 per NucleoSpin RNA L Column Mix thoroughly but gently Digest with rDNase Apply 250 uL DNase reaction mixture directly onto the center of the silica membrane Incubate at room temperature for 15 min Wash and dry silica membrane Add 2 6 mL Buffer RA2 to the NucleoSpin RNAL Column Incubate at room temperature for 2 min Centrifuge for 3 min at 4 500 x g Discard flow through and place the column back into the Collection Tube Buffer RA2 will inactivate the rDNase Add 2 6 mL Buffer RA3 to the NucleoSpin RNAL Column Centrifuge for 3 min at 4 500 x g The flow through has not to be discarded in this step Leave the NucleoSpin RNA L Column in the Collection Tube Add 2 6 mL Buffer RA3 to the NucleoSpin RNAL Column Centrifuge for 5 min at 4 500 x g to dry the membrane completely Place the column into a fresh Collection Tube 15 mL supplied q amp 250 uL rDNase
38. ozen powder to an appropriate aliquot of Buffer RA1 containing reducing agent e g B mercaptoethanol DTT or TCEP and mix immediately The broken up tissue must then be homogenized with a NucleoSpin Filter Filter L included in the kit or by passing 5 times through a 0 9 mm syringe needle Thawing of undisrupted animal tissue should be exclusively done in the presence of Buffer RA1 during simultaneous mechanical disruption for example with a rotor stator homogenizer This ensures that the RNA is not degraded by RNases before the preparation has started The spinning rotor disrupts and simultaneously homogenizes the sample by mechanical shearing of DNA within seconds up to minutes MACHEREY NAGEL 03 2011 Rev 13 13 Total RNA isolation homogenization time depends on sample Take care to keep the rotor tip submerged in order to avoid excess foaming Select a suitably sized homogenizer 5 7 mm diameter rotors can be used for homogenization in microcentrifuge tubes IEECCMEWEUD BEIGE have to be incubated in lysozyme or lyticase zymolase solutions respectively see support protocols in section 5 By this treatment the robust cell walls of these organisms are digested or at least weakened which is essential for effective cell lysis by Buffer RA1 For microorganisms with extremely resistant cell walls like some Gram positive bacterial strains it may be necessary to optimize the conditions of the treatment with lytic enz
39. pin RNA kits allow purification of pure RNA with an Aggo Ago ratio generally exceeding 1 9 measured in TE buffer pH 7 5 Even biological samples which are sometimes difficult to process will yield high quality RNA Such samples are for example mouse tissue liver brain different tumor cell lines Streptococci and Actinobacillus pleuropneumoniae The isolated RNA is ready to use for applications like reverse transcriptase PCR RT PCR primer extension or RNase protection assays RNA isolated with NucleoSpin RNA kits is of high integrity RIN RNA Integrity Number of RNA isolated from fresh high quality sample material e g eukaryotic cells or fresh mouse liver generally exceeds 9 0 However RNA integrity strongly depends on the sample quality RNA integrity was examined using the Agilent 2100 Bioanalyzer in conjunction with the RNA 6000 Nano or Pico assay The amount of DNA contamination is significantly reduced during on column digestion with rDNase Anyhow in very sensitive applications it might be possible to detect traces of DNA The NucleoSpin RNA II on column DNA removal is tested with the following procedure One million HeLa cells are subjected to RNA isolation according to the protocol RNA eluate is used as template for PCR detection of a 1 kb fragment in a 30 cycle reaction Generally no PCR fragment is obtained if the DNase is applied while a strong PCR fragment may be obtained if the DNase digestion is omitted The
40. quality or yield continued Sample material Sample material not stored properly Whenever possible use fresh material If this is not possible flash freeze the samples in liquid N Samples should always be kept at 70 C Never allow tissues to thaw before addition of Buffer RA1 Perform disruption of samples in liquid No Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters Filters L for easy homogenization of disrupted starting material Low Asgo Aas0 ratio Carry over of guanidinium thiocyanate Carefully load the lysate to the NucleoSpin RNA II Column and try to avoid a contamination of the upper part of the column and the column lid Make sure that residual Wash Buffer RA2 is washed away with Wash Buffer RA3 This may be done by applying Buffer RAS to the inner rim of the column Clogged NucleoSpin Column Poor RNA quality or yield Sample material Too much starting material used Overloading may lead to decreased overall yield Reduce amount of sample material or use larger volume of Buffer RA1 Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters Filters L for easy homogenization of disrupted starting material Contamination rDNase not active Reconstitute and store lyophilized rDNase according to instructions given in section 3 DNa
41. se solution not properly applied N Pipette rDN lution directl to th t f the sili genomic DNA ipette rDNase solution directly onto the center of the silica membrane Too much cell material used Reduce quantity of cells or tissue used 42 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation DNA detection system too sensitive The amount of DNA contamination is effectively reduced during the on column digestion with rDNase Anyhow it can not be guaranteed that the purified RNA is 10096 free of DNA therefore in very sensitive applications it might still be possible to detect DNA The NucleoSpin RNA II Plant System is checked by the following procedure One million HeLa cells are subjected to RNA isolation according to the protocol RNA eluate is used as template for PCR detection Contamination of a 1 kb fragment in a 30 cycle reaction Generally no PCR of RNA with product is obtained while skipping the DNase digest usually leads to positive PCR results The probability of DNA detection with PCR increases with the number of DNA copies per preparation single copy target plastidial mitochondrial target plasmid transfected into cells decreasing of PCR amplicon size genomic DNA continued Use larger PCR targets e g gt 500 bp or intron spanning primers if possible Use support protocol 5 11 for subsequent rDNase digestion in solution Carry over of ethanol or salt Do not let the flow throug
42. srupt up to 30 mg of tissue for sample amounts see section 2 2 for homogenization methods see section A Disrupt 2 9 sample Up to 5 x 109 eukaryotic cultured cells can be collected by centrifugation and lysed by addition of Buffer RA1 directly 2 Lysecells Add 350 uL Buffer RA1 and 3 5 pL B mercaptoethanol B ME to the cell pellet or to ground tissue and vortex vigorously 350 uL RA1 For appropriate sample and lysis buffer amounts see section 3 5 uL B ME 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 3 Filtrate lysate Reduce viscosity and clear the lysate by filtration through NucleoSpin Filter violet ring Place NucleoSpin Filter in a Collection Tube 2 mL apply the mixture and centrifuge for 1 min at 11 000 x g The lysate may be passed alternatively 2 5 times through a 0 9 mm needle 20 gauge fitted to a syringe 11 000 x g 1 min In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 1 5 mL microcentrifuge tube not supplied MACHEREY NAGEL 03 2011 Rev 13 19 NucleoSpin RNA II Important To process higher amounts of cells gt 1 x 109 or tissue gt 10 mg the lysate should first be homo genized using the 0 9 mm needle 20 gauge followed by filtration through NucleoSpin Filters
43. st time Experienced users however may refer to the Protocol at a glance instead The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com MACHEREY NAGEL 03 2011 Rev 13 T Total RNA isolation 2 Product description 2 1 The basic principle One of the most important aspects in the isolation of RNA is to prevent degradation of the RNA during the isolation procedure With the NucleoSpin RNA methods cells are lysed by incubation in a solution containing large amounts of chaotropic ions This lysis buffer immediately inactivates RNases which are present in virtually all biological materials and creates appropriate binding conditions which favor adsorption of RNA to the silica membrane Contaminating DNA which is also bound to the silica membrane is removed by an rDNase solution which is directly applied onto the silica membrane during the preparation RNase free rDNase is supplied with the kit Simple washing steps with two different buffers remove salts metabolites and macromolecular cellular components Pure RNA is finally eluted under low ionic strength conditions with RNase free H2O supplied The RNA preparation using NucleoSpin RNA kits can be performed at room temperature The eluate however should be treated with care because RNA is very sensitive to trace
44. standard protocol see sections 5 1 5 6 Cell lysis of adherent growing cells in a culture dish Completely aspirate cell culture medium and continue immediately with the addition of Lysis Buffer RA1 to the cell culture dish Avoid incomplete removal of the cell culture medium in order to allow full lysis activity of the lysis buffer To trypsinize adherent growing cells Aspirate cell culture medium and add an equal amount of PBS in order to wash the cells Aspirate PBS Add 0 1 0 3 trypsin in PBS and incubate for an appropriate time to detach the cells from the dish surface After cell detachment add medium transfer cells to an appropriate tube not supplied and pellet by centrifugation for 5 min at 300 x g Remove supernatant and continue with the addition of lysis buffer to the cell pellet are often solid and must therefore be broken up mechanically as well as lysed Depending on the disruption method the viscosity of the lysed sample has to be reduced further for optimal results It is essential for efficient RNA preparation that all the RNA contained in the sample is released from the cells by disruption and that the viscosity of the sample is reduced by homogenization The most commonly used technique for disruption of animal tissues is grinding with a pestle and mortar Grind the sample to a fine powder in the presence of liquid No Take care that the sample does not thaw during or after grinding or weighing and add the fr
45. vailable as additional solution to adjust RNA binding conditions in the lysate Check that reducing agent B ME DTT or TCEP is available Before starting any NucleoSpin RNA II protocol prepare the following rDNase RNase free Add indicated volume of RNase free H O see table below to the rDNase vial and incubate for 1 min at room temperature Gently swirl the vials to completely dissolve the rDNase Be careful not to mix rDNase vigorously as rDNase is sensitive to mechanical agitation Dispense into aliquots and store at 20 C The frozen working solution is stable for 6 months Do not freeze thaw the aliquots more than three times Be careful when opening the vial as some particles of the lyophilisate may be attached to the lid Wash Buffer RA3 Add the indicated volume of 96 100 ethanol see table below to Buffer RA3 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer RA3 at room temperature 18 25 C for up to one year MACHEREY NAGEL 03 2011 Rev 13 15 Total RNA isolation NucleoSpin RNA II 10 preps 20 preps 50 preps 250 preps REF 740955 10 740955 20 740955 50 740955 250 Wash 5mL 5mL 12 5 mL 3x25mL Buffer RA3 Add 20 mL Add 20 mL Add 50 mL Add 100 mL Concentrate ethanol ethanol ethanol ethanol to each vial rDNase 1 vial size C 1 vial size C 1 vial size D 5 vials size D RNase free Add 230 uL Add 230 uL Add 540 uL Add 540 uL lyophilized RNase
46. ymes or the cultivation conditions After lysis homogenization is achieved by the use of a NucleoSpin Filter or the syringe needle method 2 4 Elution procedures It is possible to adapt elution method and volume of water used for the subsequent application of interest In addition to the standard method described in the individual protocols recovery rate about 70 90 96 there are several modifications possible High yield Perform two elution steps with the volume indicated in the individual protocol About 90 100 of bound nucleic acid will be eluted High yield and high concentration Elute with the standard elution volume and apply the eluate once more onto the column for reelution Eluted RNA should immediately be put and always kept on ice for optimal stability because almost omnipresent RNases general lab ware fingerprints dust will degrade RNA For short term storage freeze at 20 C for long term storage freeze at 70 C 14 MACHEREY NAGEL 03 2011 Rev 13 Total RNA isolation 3 Storage conditions and preparation of working solutions Attention Buffers RA1 RA2 and MDB contain guanidine thiocyanate Wear gloves and goggles Store lyophilized rDNase RNase free at 4 C on arrival stable up to 1 year All other kit components should be stored at room temperature 18 25 C and are stable up to one year Storage at lower temperatures may cause precipitation of salts Check that 7096 ethanol is a

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