Home

RNeasy Fibrous Tissue Handbook

1. m Operate the TissueLyser for 3 min at 20 Hz The time depends on the tissue being processed and can be extended until the tissue is completely homogenized 22 RNeasy Fibrous Tissue Handbook 11 2006 m Rearrange the tubes so that the outermost tubes are innermost and the inner most tubes are outermost Operate the TissueLyser for another 3 min at 20 Hz Rearranging the tubes allows even homogenization Carefully pipet the lysates into new microcentrifuge tubes not supplied Adjust the volume to 2 ml with Buffer RLT Proceed to step 5 Do not reuse the stainless steel beads 5 Add 4 ml RNase free water to the lysate Then add 65 pl proteinase K solution and mix thoroughly by pipetting 6 Incubate at 55 C for 20 min 7 Centrifuge at 20 25 C for 5 min at 3000 5000 x g A small pellet of tissue debris will form sometimes accompanied by a thin layer or film on top of the supernatant r Z y fe n x om ke c nn wn n c 8 Pipet the supernatant approximately 6 ml into a new 10 15 ml centrifuge tube not supplied Avoid transferring any of the pellet If this is unavoidable a small amount of pelleted debris may be carried over without affecting the RNeasy procedure Hold the pipet tip under the thin layer or film on top of the supernatant if present This layer will usually adhere to the outside of the pipet tip and should not be transferred 9 Add 0 5 volumes usually 3 ml of ethanol 96 100
2. to the cleared lysate Mix well by pipetting Do not centrifuge Precipitates may be visible after addition of ethanol This does not affect the procedure 10 Transfer 3 ml of the sample including any precipitate that may have formed to an RNeasy Midi spin column placed in a 15 ml collection tube Close the lid gently and centrifuge at 20 25 C for 5 min at 3000 5000 x g Discard the flow through Reuse the collection tube in step 11 11 Repeat step 10 twice the first time using 3 ml of the sample and the second time using the remainder of the sample about 3 ml Discard the flow through Reuse the collection tube in step 12 12 Add 2 ml Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge at 20 25 C for 5 min at 3000 5000 x g to wash the membrane Discard the flow through Reuse the collection tube in step 15 Flow through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information RNeasy Fibrous Tissue Handbook 11 2006 23 Optional If on column DNase digestion is not desired add 4 ml Buffer RW1 instead centrifuge for 5 min at 3000 5000 x g and discard the flow through but not the collection tube Proceed to step 16 13 Add 20 pl DNase I stock solution to 140 pl Buffer RDD Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube Note DNase is especially sensitive to physical denatu
3. oO RNeasy Fibrous Tissue Handbook 11 2006 19 v wn Ane D a au oo 2 z 2 wn 5 oO Z 4 Protocol Purification of Total RNA Using the RNeasy Fibrous Tissue Midi Kit Determining the correct amount of starting material It is essential to use the correct amount of tissue in order to obtain optimal RNA yield and purity With the RNeasy Fibrous Tissue Midi Kit a maximum of 250 mg tissue can generally be processed For most tissues the RNA binding capacity of the RNeasy spin column and the lysing capacity of Buffer RLT and proteinase K will not be exceeded by this amount Average RNA yields from various tissues are given in Table 2 page 12 If there is no information about the nature of your starting material we recommend starting with no more than 100 mg tissue Depending on the RNA yield from the tissue sample it may be possible to use up to 250 mg tissue in subsequent preparations Do not overload the RNeasy spin column as this will significantly reduce RNA yield and quality Weighing tissue is the most accurate way to quantify the amount of starting material As a guide a 5 mm cube 125 mm of most animal tissues weighs 150 175 mg Important points before starting E If using RNeasy Fibrous Tissue Kits for the first time read Important Notes page 11 If working with RNA for the first time read Appendix A page 30 E If using the TissueRuptor ensure that you are familiar with operating it
4. so that the RNA binding capacity of the RNeasy spin column is not exceeded When processing samples containing low amounts of RNA the maximum amount of starting material shown in Table 1 can be used However even though the RNA binding capacity of the RNeasy spin column is not reached the maximum amount of starting material must not be exceeded Otherwise lysis will be incomplete and cellular debris may interfere with the binding of RNA to the RNeasy spin column membrane resulting in lower RNA yield and purity More information on using the correct amount of starting material is given in the protocols Table 2 shows expected RNA yields from various sources Table 1 RNeasy Spin Column Specifications RNeasy Mini spin RNeasy Midi spin Specification column column Maximum binding capacity 100 pg RNA 1 mg RNA Maximum loading volume 700 pl 4 ml RNA size distribution RNA gt 200 RNA gt 200 nucleotides nucleotides Minimum elution volume 30 pl 150 pl Maximum amount of starting tissue lt 30 mg 20 250 mg Note If the binding capacity of the RNeasy spin column is exceeded RNA yields will not be consistent and may be reduced If lysis of the starting material is incomplete RNA yields will be lower than expected even if the binding capacity of the RNeasy spin column is not exceeded RNeasy Fibrous Tissue Handbook 11 2006 11 Table 2 Typical Yields of Total RNA with RNeasy Fibrous Tissue Kits Mouse rat tissue 10 m
5. 75842 Midi Kit 10 Collection Tubes QIAzol Lysis Reagent RNase Free Reagents and Buffers Larger kit size available see www giagen com products PCR t Larger kit sizes and format available see www giagen com RNA 38 RNeasy Fibrous Tissue Handbook 11 2006 Ordering Information Product Contents Cat no RNeasy 96 Universal Tissue Kit for high throughput RNA purification from any type of animal tissue RNeasy 96 Universal For 4 x 96 total RNA preps 74881 Tissue Kit 4 4 RNeasy 96 Plates Collection Microtubes Elution Microtubes CL Caps S Blocks AirPore Tape Sheets QIAzol Lysis Reagent RNase Free Reagents and Buffers miRNeasy Kits for purification of microRNA and total RNA from a wide range of animal tissues and cells miRNeasy Mini Kit 50 For 50 preps 50 RNeasy Mini Spin 217004 Columns Collection Tubes 1 5 ml and 2 ml QIAzol Lysis Reagent RNase Free Reagents and Buffers miRNeasy 96 Kit 4 For 4 x 96 preps 4 RNeasy 217061 96 plates Collection Microtubes racked Elution Microtubes CL Caps S Blocks AirPore Tape Sheets QIAzol Lysis Reagent RNase Free Reagents and Buffers RNAlater RNA Stabilization Reagent RNAlater TissueProtect Tubes the TissueRuptor the Tissuelyser the RNeasy Midi Kit the RNeasy Plus Mini Kit RNeasy Lipid Tissue Kits and QuantiTect Kits and Assays are intended for research use No claim or representation is intended to provide information for the diagnosi
6. Tris Cl pH 7 0 1 50 dilution Measure absorbance of diluted sample in a 1 ml cuvette RNase free Mic Concentration of RNA sample 44 pg ml x Azo x dilution factor 44 pg ml x 0 2 x 50 440 pg ml 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 32 RNeasy Fibrous Tissue Handbook 11 2006 Total amount concentration x volume in milliliters 440 pg ml x 0 1 ml 44 pg of RNA Purity of RNA The ratio of the readings at 260 nm and 280 nm Az6o A290 provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the A260 Azs ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Ap6o Azgo ratio can vary greatly Lower pH results in a lower Apzgo Azgo ratio and reduced sensitivity to protein contamination For accurate values we recommend measuring absorbance in 10 mM Tris Cl pH 7 5 Pure RNA has an Aj6o Azgo ratio of 1 9 2 1 in 10 mM Tris Cl pH 7 5 Always be sure to calibrate the spectrophotometer with the same solution used for dilution For determination of RNA concentration however we recommend dilution of the sample in a buffer with neutral pH since the relationship between absorbance and concentration Azs reading of 1 44 pg ml RNA is based
7. be carried out as quickly as possible 4 Disrupt the tissue and homogenize the lysate using either the TissueRupter follow step 4a or Tissuelyser follow step 4b See Disrupting and homogenizing starting material page 12 for more details on disruption and homogenization Note Ensure that B ME is added to Buffer RLT before use see Things to do before starting Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column Homogenization with the TissueRupter or TissueLyser generally results in higher RNA yields than with other methods Aa Disruption and homogenization using the TissueRuptor m Place the tissue in a suitably sized vessel Add 2 ml Buffer RLT Generally round bottomed tubes allow more efficient disruption and homog enization than conical bottomed tubes m Place the tip of the disposable probe into the vessel and operate the TissueRuptor at full speed until the lysate is uniformly homogeneous usually 45 60 s Proceed to step 5 Note To avoid damage to the TissueRuptor and disposable probe during operation make sure the tip of the probe remains submerged in the buffer Ab Disruption and homogenization using the TissueLyser m Place the tissues in the tubes prepared in step 2 m If the tubes were stored on dry ice place them at room temperature Then immediately add 1 ml Buffer RLT per tube m Place the tubes in the Tissuelyser Adapter Set 2 x 24
8. by referring to the TissueRuptor User Manual and TissueRuptor Handbook E If using the TissueLlyser ensure that you are familiar with operating it by referring to the operating instructions and Tissuelyser Handbook MH Since the RNase inactivating Buffer RLT must be diluted to permit proteinase K digestion this protocol should not be used for tissues rich in RNases such as pancreas or intestine E Fresh frozen or RNAlater stabilized tissues can be used If freezing tissues flash freeze in liquid nitrogen and immediately transfer to 70 C where they can be stored for several months Do not allow tissues to thaw during weighing or handling prior to disruption in Buffer RLT Homogenized tissue lysates from step 4 can also be stored at 70 C for several months Incubate frozen lysates at 37 C in a water bath until completely thawed and salts are dissolved before continuing with step 5 Avoid prolonged incubation which may compromise RNA integrity E Do not vortex reconstituted DNase I DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube E Buffer RIT may form a precipitate upon storage If necessary redissolve by warming and then place at room temperature 15 25 C 20 RNeasy Fibrous Tissue Handbook 11 2006 Buffer RIT and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 6 for safety inf
9. collection microtubes 1 2 ml 960 in strips of 8 100 V 50 60 Hz for Japan t 120 V 50 60 Hz for North America 220 240 V 50 60 Hz for Europe and Australia RNeasy Fibrous Tissue Handbook 11 2006 37 Ordering Information Product Contents Cat no Related products QuantiTect Reverse Transcription Kit for fast cDNA synthesis for sensitive real time two step RT PCR QuantiTect Reverse For 50 x 20 pl reactions gDNA 205311 Transcription Kit 50 Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix and RNase Free Water RNeasy Kits for purification of total RNA from animal cells or tissues or yeast RNeasy Mini Kit 50 50 RNeasy Mini Spin Columns 74104 Collection Tubes RNase Free Reagents and Buffers RNeasy Midi Kit 10 10 RNeasy Midi Spin Columns 75142 Collection Tubes RNase Free Reagents and Buffers RNeasy Plus Mini Kit for purification of total RNA from cultured cells and tissues using gDNA Eliminator columns RNeasy Plus Mini Kit 50 50 RNeasy Mini Spin Columns 74134 50 gDNA Eliminator Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers RNeasy Lipid Tissue Kits for purification of total RNA from all types of animal tissue including fatty tissues RNeasy Lipid Tissue 50 RNeasy Mini Spin Columns 74804 Mini Kit 50 Collection Tubes QIAzol Lysis Reagent RNase Free Reagents and Buffers RNeasy Lipid Tissue 10 RNeasy Midi Spin Columns
10. dry ice For users of RNeasy Fibrous Tissue Mini Kit E 1 5 ml or 2 ml microcentrifuge tubes MH Microcentrifuge with rotor for 2 ml tubes For users of RNeasy Fibrous Tissue Midi Kit M 10 15 ml centrifuge tubes MH Laboratory centrifuge capable of 3000 5000 x g e g Centrifuge 4 15C or Centrifuge 4K 15C from QIAGEN please inquire with a swinging bucket rotor for 15 ml centrifuge tubest Do not use denatured alcohol which contains other substances such as methanol or methylethylketone t A maximum speed of 3500 5000 rpm corresponds to 3000 5000 x g for most rotors RNeasy Midi spin columns fit into 15 ml collection tubes supplied with the kit These fit into the rotor of almost every standard laboratory centrifuge available In the unlikely event that the tubes do not fit RNeasy Midi spin columns can also be inserted into different 12 15 ml RNase free glass or polypropylene tubes 10 RNeasy Fibrous Tissue Handbook 11 2006 Important Notes Determining the amount of starting material It is essential to use the correct amount of starting material in order to obtain optimal RNA yield and purity The maximum amount that can be used is determined by E The type of tissue and its RNA content MH The volume of Buffer RLT required for efficient lysis E The RNA binding capacity of the RNeasy spin column When processing samples containing high amounts of RNA less than the maximum amount of starting material shown in Table 1 should be used
11. fiber rich tissues in 96 well format For ordering information see page 39 Ifthe TissueRuptor TissueLyser or other similar instrument is not available contact QIAGEN Technical Services for an alternative method of disruption and homogenization RNeasy Fibrous Tissue Handbook 11 2006 13 o a a o LL a 5 7 Z S iE Protocol Purification of Total RNA Using the RNeasy Fibrous Tissue Mini Kit Determining the correct amount of starting material It is essential to use the correct amount of tissue in order to obtain optimal RNA yield and purity With the RNeasy Fibrous Tissue Mini Kit a maximum of 30 mg tissue can generally be processed For most tissues the RNA binding capacity of the RNeasy spin column and the lysing capacity of Buffer RLT and proteinase K will not be exceeded by this amount Average RNA yields from various tissues are given in Table 2 page 12 If there is no information about the nature of your starting material we recommend starting with no more than 10 mg tissue Depending on the RNA yield from the tissue sample it may be possible to use up to 30 mg tissue in subsequent preparations Do not overload the RNeasy spin column as this will significantly reduce RNA yield and quality Weighing tissue is the most accurate way to quantify the amount of starting material As a guide a 3 mm cube 27 mm of most animal tissues weighs 25 35 mg Important points befo
12. for 10 min 5 Centrifuge at 10 000 x g for 3 min 6 Transfer supernatant to new tube add 0 5 volumes of 96 100 ethanol and mix Do not centrifuge 7 Transfer sample to RNeasy column in 2 ml tube Close lid centrifuge for 15 s at gt 8000 x g and discard flow through 8 Add 350 pl Buffer RW1 to RNeasy column Close lid centrifuge for 15 s at 28000 x g and discard flow through 9 Mix 10 pl DNase stock solution with 70 pl Buffer RDD add to RNeasy membrane and incubate for 15 min at 20 30 C 10 Add 350 pl Buffer RW1 to RNeasy column Close lid centrifuge for 15 s at 28000 x g and discard flow through 11 Add 500 pl Buffer RPE to RNeasy column Close lid centrifuge for 15 s at 28000 x g and discard flow through 12 Add 500 pl Buffer RPE to RNeasy column Close lid and centrifuge for 2 min at 28000 x g 13 Optional Place RNeasy column in new 2 ml tube close lid and centrifuge at full speed for 1 min 14 Place RNeasy column in new 1 5 ml tube Add 30 50 pl RNase free water close lid and centrifuge for 1 min at gt 8000 x g Optional Repeat elution with another volume of water or with RNA eluate Bench Protocol RNA Purification Using seses the RNeasy Fibrous Tissue Midi Kit QIAGEN Note Before using this bench protocol you should be completely familiar with the safety information and protocols in the RNeasy Fibrous Tissue Handbook Important points before starting M Except for steps 4 and 9 perform the
13. months Thawed aliquots can be stored at 2 8 C for up to 6 weeks Do not refreeze the aliquots after thawing Procedure 1 2 Heat a water bath or heating block to 55 C for proteinase K digestion in step 6 If using the TissueLyser add one stainless steel bead 5 mm mean diameter per 2 ml microcentrifuge tube not supplied If working with tissues that are not stabilized in RNAlafer RNA Stabilization Reagent place the tubes on dry ice Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue Do not use more than 250 mg Proceed immediately to step 4 RNeasy Fibrous Tissue Handbook 11 2006 21 A Z y fe a x ay o S e c a a a c oO o wn A A am au b a Peire z 2 ee wn 5 oO Z 4 If using the Tissuelyser we recommend no more than 150 mg tissue for optimal lysis Depending on the organism and the type of tissue up to 250 mg tissue may be possible Weighing tissue is the most accurate way to determine the amount If the tissue sample was stored in RNAlatfer RNA Stabilization Reagent remove it from the reagent using forceps and be sure to remove any crystals that may have formed RNA in harvested tissues is not protected until the tissues are treated with RNAlater RNA Stabilization Reagent flash frozen or disrupted and homogenized in step 4 Frozen tissues should not be allowed to thaw during handling The relevant procedures should
14. temperature Then immediately add 300 pl Buffer RLT per tube m Place the tubes in the Tissuelyser Adapter Set 2 x 24 m Operate the TissueLyser for 2 min at 20 Hz The time depends on the tissue being processed and can be extended until the tissue is completely homogenized m Rearrange the tubes so that the outermost tubes are innermost and the inner most tubes are outermost Operate the TissueLyser for another 2 min at 20 Hz Rearranging the tubes allows even homogenization RNeasy Fibrous Tissue Handbook 11 2006 m Carefully pipet the lysates into new microcentrifuge tubes not supplied Proceed to step 5 Do not reuse the stainless steel beads 5 Add 590 pl RNase free water to the lysate Then add 10 pl proteinase K solution and mix thoroughly by pipetting 6 Incubate at 55 C for 10 min 7 Centrifuge at 20 25 C for 3 min at 10 000 x g A small pellet of tissue debris will form sometimes accompanied by a thin layer or film on top of the supernatant r z G Q N x lt om ke i wn wn N 0 8 Pipet the supernatant approximately 900 pl into a new 1 5 ml or 2 ml microcentrifuge tube not supplied Avoid transferring any of the pellet If this is unavoidable a small amount of pelleted debris may be carried over without affecting the RNeasy procedure Hold the pipet tip under the thin layer or film on top of the supernatant if present This layer will usually adhere to the outside of th
15. 0 pl final volume C3 Incubate on the benchtop 20 25 C for 10 min C4 Clean up the RNA using the A RNeasy Mini Kit or RNeasy Midi Kit see ordering information page 38 RNeasy Fibrous Tissue Handbook 11 2006 35 Ordering Information Product Contents Cat no 74704 RNeasy Fibrous Tissue Mini Kit 50 RNeasy Fibrous Tissue Midi Kit 10 Accessories RNAlater RNA Stabilization Reagent 50 ml RNAlater RNA Stabilization Reagent 250 ml RNAlater TissueProtect Tubes 50 x 1 5 ml RNAlater TissueProtect Tubes 20 x 5 ml TissueRuptor TissueRuptor Disposable Probes 25 50 RNeasy Mini Spin Columns Collection Tubes Proteinase K RNase Free DNase RNase Free Reagents and Buffers 10 RNeasy Midi Spin Columns Collection Tubes Proteinase K RNase Free DNase RNase Free Reagents and Buffers For stabilization of RNA in 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent For stabilization of RNA in 125 x 200 mg tissue samples 250 ml RNAlater RNA Stabilization Reagent For stabilization of RNA in 50 x 150 mg tissue samples 50 screw top tubes containing 1 5 ml RNA ater RNA Stabilization Reagent each For stabilization of RNA in 20 x 500 mg tissue samples 20 screw top tubes containing 5 ml RNAlater RNA Stabilization Reagent each Handheld rotor stator homogenizer 5 TissueRuptor Disposable Probes 25 nonsterile plastic disposable probes for use with the Ti
16. 26 36 37 Proteinase K Contains proteinase K sensitizer irritant Risk and safety phrases R36 37 38 42 43 S23 24 26 36 37 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 R10 Flammable R20 21 22 Harmful by inhalation in contact with skin and if swallowed R32 Contact with acids liberates very toxic gas R36 37 38 Irritating to eyes respiratory system and skin R42 43 May cause sensitization by inhalation and skin contact S13 Keep away from food drink and animal feedingstuffs S22 Do not breathe dust S23 Do not breathe vapor 24 Avoid contact with skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 Wear suitable protective clothing S36 37 Wear suitable protective clothing and gloves S46 If swallowed seek medical advice immediately and show the container or label RNeasy Fibrous Tissue Handbook 11 2006 7 Introduction RNeasy Fibrous Tissue Kits are designed for optimal lysis of fiber rich tissues and purification of high quality total RNA QIAGEN provides a wide range of other kits for purification of total RNA from different sample sources visit www giagen com RNA Principle and procedure Total RNA purification from fibrous tissues such as skeletal muscle heart and aorta tissue can be difficult due to the
17. 3 5547 0811 Fax 03 5547 0818 Technical 03 5547 0811 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN WWW QIAGEN COM ari Please tear off here Bench Protocol RNA Purification Using 00000 the RNeasy Fibrous Tissue Mini Kit QIAGEN Note Before using this bench protocol you should be completely familiar with the safety information and protocols in the RNeasy Fibrous Tissue Handbook Important points before starting M Except for step 4 and 9 perform the procedure at 15 25 C Work quickly E Perform centrifugation at 20 25 C E Redissolve any precipitate in Buffer RLT by warming Add 8 ME before use E Before using Buffer RPE for the first time ensure ethanol is added E Prepare DNase stock solution Procedure 1 Heat water bath or heating block to 55 C 2 Disrupt and homogenize lt 30 mg tissue in 300 pl Buffer RLT using the TissueRuptor or TissueLyser 3 Add 590 pl RNase free water then 10 pl Proteinase K and mix 4 Incubate at 55 C
18. 30 80 40 Tel 372 7301321 Tel 603 8070 3101 Tel Fax 02 5022 1336 Fax 386 1 830 80 70 Fax 972 7304310 Fax 603 8070 5101 Email bio cons cdicon sk 386 1 830 80 63 E mail quantum quantum ee E mail biolabs tm net my Shes E mail info mediline si Greece oe MEDIN des F BioAnalytica S A no Tel 01 830 80 40 Brazil ij Quimica Valaner S A de C V ae Tel 210 40 03 18 Fox 101 830 8070 Uniscience do Brasil A Tel 55 55 25 57 25 an Fax 210 646 27 48 016308043 Tel 011 3622 2320 Email bioanalyt hol gr Fax 55 55 25 56 25 _ 01 830 8063 Fax 011 3622 2323 Email ventas valaner com Email info mediline si E mail info uniscience com Hong Kong SAR 7 Gene Company Limited New Zealand Sa Ga Biotechnol Chile Tel 852 2896 6283 Biolab Ltd Mal e EN SaRY Biosonda SA Fax 852 2515 9371 Tel 09 980 6700 Py oer Tel 562 209 6770 E mail info genehk com 0800 933 966 5 ae rea 6717734 Fax 562 274 5462 i Fax 09 980 6788 oan k b Egal ventas biosonda cl Genetimes Technology International Email E mail info scb co za Holding Lid biosciences nzl biolabgroup com Spain China Tel 852 2385 2818 pal in Scientifi Fax 852 2385 1308 IZASA S A Eastwin Scientific Inc a Oman Tel 93 902 20 30 90 Order 86 400 8182168 oe eer hk Al Mazouri Medical amp Chemical Fax 93 902 22 33 66 Tel 86 10 51663168 Sgn nd gee ee Somy Supplies E mail consultasbiotec izasa es Fax 86 1082898283 Hingis Tel 971 4 266 1272 E mail laborder east
19. November 2006 RNeasy Fibrous Tissue Handbook RNeasy Fibrous Tissue Mini Kit RNeasy Fibrous Tissue Midi Kit For purification of total RNA from heart skeletal muscle aorta and other fiber rich tissues QIAGEN Trademarks QIAGEN RNeasy Quantiscript QuantiTect QIAGEN Group ABI PRISM Applera Corporation or its subsidiaries Agilent Agilent Technologies Inc LightCycler Roche Group SYBR Molecular Probes Inc RNAlater is a trademark of AMBION Inc Austin Texas and is covered by various U S and foreign patents QIAzol Lysis Reagent is a subject of US Patent No 5 346 994 and foreign equivalents 2002 2006 QIAGEN all rights reserved Contents Kit Contents 4 Shipping and Storage A Quality Control 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 6 Safety Information 6 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 Determining the amount of starting material 11 Handling and storing starting material 12 Disrupting and homogenizing starting material 12 Protocols Oo Purification of Total RNA Using the RNeasy Fibrous Tissue Mini Kit 14 a Purification of Total RNA Using the RNeasy Fibrous Tissue Midi Kit 20 Troubleshooting Guide 26 Appendix A General Remarks on Handling RNA 30 Appendix B Storage Quantification and Determination of Quality of RNA 32 Appendix C DNase Digesti
20. abundance of contractile proteins connective tissue and collagen RNeasy Fibrous Tissue Kits are supplied with proteinase K which removes these proteins Tissue samples are first lysed in Buffer RLT and then diluted before being treated with proteinase K Debris is pelleted by centrifugation and the supernatant is removed The supernatant is mixed with ethanol and then centrifuged through an RNeasy spin column where RNA binds to the silica membrane Traces of DNA that may copurify with the RNA are removed by DNase treatment on the silica membrane DNase and any contaminants are efficiently washed away and high quality total RNA is eluted in RNase free water see flowchart next page RNeasy Fibrous Tissue Kits are available in two formats E RNeasy Fibrous Tissue Mini Kit for RNA purification from up to 30 mg tissue using RNeasy Mini spin columns M RNeasy Fibrous Tissue Midi Kit for RNA purification from up to 250 mg tissue using RNeasy Midi spin columns With RNeasy Fibrous Tissue Kits all RNA molecules longer than 200 nucleotides are purified The procedure provides an enrichment for mRNA since most RNAs lt 200 nucleotides such as 5 85 rRNA 5S rRNA and tRNAs which together comprise 15 20 of total RNA are selectively excluded The size distribution of the purified RNA is comparable to that obtained by centrifugation through a CsCl cushion where small RNAs do not sediment efficiently For purification of small RNA including m
21. ase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Do not vortex 14 Add the DNase incubation mix 80 pl directly to the RNeasy spin column membrane and place on the benchtop 20 30 C for 15 min S iE Note Be sure to add the DNase incubation mix directly to the RNeasy spin column membrane DNase digestion will be incomplete if part of the mix sticks to the walls or the O ring of the spin column 15 Add 350 pl Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm at 20 25 C Discard the flow through o a n o LL a 5 Z Reuse the collection tube in step 16 16 Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge at 20 25 C for 15 s at gt 8000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step 17 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting 17 Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge at 20 25 C for 2 min at gt 8000 x g 210 000 rpm to wash the membrane The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with down stream reac
22. cal Tel 57 4 2519037 l i Fax 98 21 88214107 Fax 071 798 58 53 Supplies Fax 57 4 2516555 sa APE AI a Ge IN PP E mail E mail info biorain com E mail info syngen pl Tel 971 4 266 1272 gerencia gentechcolombia com Israel 7 i Ee ei oea Cea soporte gentechcolombia com Eldan Electronic Instruments Co Ltd AST PORTUGAL IDA we ATTN LAB DIVISION Tel 972 3 937 1133 Tel y haji al Croatia Fak 4972 3937 1121 el 21 424 7312 E mail shaji almaz net ae INEL Medicinska Tehnika d o 0 Email bio eldan biz Fax 3 21 417 2674 i Tel 01 2984 898 E mail consultasbiotec izasa es roau i Fax 01 6520 966 Jordan PONEI care SAHOURY GROUP Gir Tel 598 2 6130442 inelmedicinskatehnika zg hinet hr Tel 962 6 4633290 111 Sedeer Medical To Gove 2 ONa2S92 Fax 962 6 4633290 110 Tel 974 488 5218 Email bionova internet com uy Cyprus E mail info sahoury com Fax 974 488 1988 Scientronics Ltd Email sedeer qatar net qa vrezi b i Tel 357 22 467880 90 Korea Tel L58212 524851 8 Foe 357 22 764614 LRS Laboratories Inc Romania j 7 Pi 58212 7616143 E mail Tel 02 924 86 97 Zyrcon Medical S R L 58212 3255838 a sarpetsas biotronics com cy Fax 02 924 86 96 Tel 40 21 2245607 F E 7615945 Email webmaster lrslab co kr Fax 40 21 2245608 OR X Czech Republic i Email E mail ventas saixx com BIO CONSUIT spol s r o Philekorea Technology Inc virgil dracea zyrconmedical ro sabaventas cantv net i Tel 02 576 6540 e T
23. d design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover RNeasy Fibrous Tissue Handbook 11 2006 5 Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products If you have any questions or experience any difficulties regarding RNeasy Fibrous Tissue Kits or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more in
24. disposable probe rotates at a very high speed causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing For guidelines on using the TissueRuptor refer to the TissueRuptor Handbook For other rotor stator homogenizers refer to suppliers guidelines Disruption and homogenization using the TissueLyser In bead milling tissues can be disrupted by rapid agitation in the presence of beads and lysis buffer Disruption and simultaneous homogenization occur by the shearing and crushing action of the beads as they collide with the cells The Tissuelyser disrupts and homogenizes up to 48 tissue samples simultaneously when used in combination with the TissueLlyser Adapter Set 2 x 24 which holds 48 x 2 ml microcentrifuge tubes containing stainless steel beads of 5 mm mean diameter For guidelines on using the Tissuelyser refer to the Tissuelyser Handbook For other bead mills refer to suppliers guidelines Note Tungsten carbide beads react with Buffer RLT and must not be used to disrupt and homogenize tissues The Tissuelyser can also disrupt and homogenize up to 192 tissue samples simultaneously when used in combination with the Tissuelyser Adapter Set 2 x 96 which holds 192 x 1 2 ml microtubes containing stainless steel beads of 5 mm mean diameter In this case we recommend using the RNeasy 96 Universal Tissue Kit which provides high throughput RNA purification from all types of tissue including
25. e 2 distinct steps E Disruption Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the RNA contained in the sample Incomplete disruption results in significantly reduced RNA yields 12 RNeasy Fibrous Tissue Handbook 11 2006 MH Homogenization Homogenization is necessary to reduce the viscosity of the lysates produced by disruption Homogenization shears high molecular weight genomic DNA and other high molecular weight cellular components fo create a homogeneous lysate Incomplete homogenization results in inefficient binding of RNA to the RNeasy spin column membrane and therefore significantly reduced RNA yields Disruption and homogenization of tissue samples can be carried out rapidly and efficiently using either the TissueRuptor for processing samples individually or the Tissuelyser for processing multiple samples simultaneously Disruption and homogenization with the TissueRuptor or Tissuelyser generally results in higher RNA yields than with other methods such as disruption in liquid nitrogen using a mortar and pestle followed by homogenization using a syringe and needle Disruption and homogenization using the TissueRuptor The TissueRuptor is a rotor stator homogenizer that thoroughly disrupts and simultaneously homogenizes single tissue samples in the presence of lysis buffer in 15 90 seconds depending on the toughness and size of the sample The blade of the TissueRuptor
26. e pipet tip and should not be transferred 9 Add 0 5 volumes usually 450 pl of ethanol 96 100 to the cleared lysate Mix well by pipetting Do not centrifuge Precipitates may be visible after addition of ethanol This does not affect the procedure 10 Transfer 700 pl of the sample including any precipitate that may have formed to an RNeasy Mini spin column placed in a 2 ml collection tube Close the lid gently and centrifuge at 20 25 C for 15 s at gt 8000 x g 210 000 rpm Discard the flow through Reuse the collection tube in step 11 11 Repeat step 10 using the remainder of the sample Discard the flow through Reuse the collection tube in step 12 12 Add 350 pl Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge at 20 25 C for 15 s at gt 8000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step 15 Optional If on column DNase digestion is not desired add 700 pl Buffer RW1 instead centrifuge for 15 s at gt 8000 x g and discard the flow through but not the collection tube Proceed to step 16 Flow through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information RNeasy Fibrous Tissue Handbook 11 2006 17 13 Add 10 pl DNase stock solution to 70 pl Buffer RDD Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube Note DN
27. ee water into the vial using an RNase free needle and syringe Mix gently by inverting the vial Do not vortex For long term storage of DNase remove the stock solution from the glass vial divide it into single use aliquots and store at 20 C for up to 9 months Thawed aliquots can be stored at 2 8 C for up to 6 weeks Do not refreeze the aliquots after thawing Procedure 1 2 Heat a water bath or heating block to 55 C for proteinase K digestion in step 6 If using the TissueLyser add one stainless steel bead 5 mm mean diameter per 2 ml microcentrifuge tube not supplied If working with tissues that are not stabilized in RNAlater RNA Stabilization Reagent place the tubes on dry ice Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue Do not use more than 30 mg Proceed immediately to step 4 Weighing tissue is the most accurate way to determine the amount RNeasy Fibrous Tissue Handbook 11 2006 15 r z G Q N x lt om ke i wn wn 17 i o o a ae a o LL a 5 7 Z S iE 4a Ab If the tissue sample was stored in RNAlater RNA Stabilization Reagent remove it from the reagent using forceps and be sure to remove any crystals that may have formed RNA in harvested tissues is not protected until the tissues are treated with RNAlater RNA Stabilization Reagent flash frozen or disrupted and ho
28. el Fax 420 2 417 29 792 Fax 02 576 6541 secretariat zyrconmedical ro Vietnam E mail info bioconsult cz ia Cie hikar okr Viet Anh Instruments Co Ltd i ii Saudi Arabia Tel 84 4 51 19452 Ecuador Latvia Abdulla Fouad Holding Company Fax 84 4 51 19453 INMUNOCHEM S A C SIA JLM Tel 03 8324400 E mail VietanhHN hn vnn yn Tel 51 1 4409678 Tel 7136393 Fax 03 8346174 Fax 51 1 4223701 Fax 7136394 E mail All other countries Email inmunochem terra com pe E mail jim mednet lv sadiq omar abdulla fouad com QIAGEN GmbH Germany RNeasy Fibrous Tissue Handbook 11 2006 43 Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 021 51345678 Fax 021 51342500 Technical 021 51345678 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 3343041 1 Fax 02 33430426 Technical 800 787980 Japan Telephone 0
29. formation please call one of the QIAGEN Technical Service Departments or local distributors see back cover Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com ts msds asp where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer RLT contains guanidine thiocyanate and Buffer RW1 contains a small amount of guanidine thiocyanate Guandine salts can form highly reactive compounds when combined with bleach If liquid containing these buffers is spilt clean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite The following risk and safety phrases apply to the components of RNeasy Fibrous Tissue Kits 6 RNeasy Fibrous Tissue Handbook 11 2006 Buffer RLT Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 13 26 36 46 Buffer RW1 Contains ethanol flammable Risk phrase R10 RNase Free DNase Contains deoxyribonuclease sensitizer Risk and safety phrases R42 43 S22 24
30. from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur 18 Place the RNeasy spin column in a new 15 ml collection tube supplied Add 150 pl RNase free water directly to the RNeasy spin column membrane Close the lid gently To elute the RNA wait for 1 min and then centrifuge for 3 min at 3000 5000 x g at 20 25 C Flow through contains Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information 24 RNeasy Fibrous Tissue Handbook 11 2006 19 Repeat step 18 using another 150 pl RNase free water or using the eluate from step 18 if high RNA concentration is required Reuse the collection tube from step 18 If using the eluate from step 18 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but the final RNA concentration will be higher A Z D e a x o S e c a a a c oO RNeasy Fibrous Tissue Handbook 11 2006 25 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or molecular biology applications see back cover for contact information Comments and suggestions Clogged RNeasy spin column a Inefficient disruption and or See Disrupting and homogenizi
31. g Yield of total RNA yg Heart 8 12 Aorta 8 12 Trachea 8 12 Esophagus 8 12 Muscle 5 10 Skin A 8 Eye 8 12 Ear 10 15 Amounts can vary due to factors such as species and developmental stage Since the RNeasy procedures enrich for mRNA and other RNA species gt 200 nucleotides the total RNA yield does not include 5S rRNA tRNA and other low molecular weight RNAs which make up 15 20 of total cellular RNA Handling and storing starting material RNA in tissues is not protected after harvesting until the sample is treated with RNAlater RNA Stabilization Reagent flash frozen or disrupted and homogenized in the presence of RNase inhibiting or denaturing reagents It is therefore important that tissue samples are immediately immersed in RNAlater RNA Stabilization Reagent see RNAlater Handbook or immediately frozen in liquid nitrogen and stored at 70 C Otherwise unwanted changes in the gene expression profile will occur The procedures for tissue harvesting and RNA protection should be carried out as quickly as possible Frozen tissue samples should not be allowed to thaw during handling or weighing After disruption and homogenization in Buffer RLT lysis buffer samples can be stored at 70 C for months Disrupting and homogenizing starting material Efficient disruption and homogenization of the starting material is an absolute requirement for all total RNA purification procedures Disruption and homogenization ar
32. i Kit 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 34 RNeasy Fibrous Tissue Handbook 11 2006 Things to do before starting E Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the lyophilized DNase 1500 Kunitz units in 550 pl of the RNase free water provided To avoid loss of DNase do not open the vial Inject RNase free water into the vial using an RNase free needle and syringe Mix gently by inverting the vial Do not vortex MH For longterm storage of DNase remove the stock solution from the glass vial divide it into single use aliquots and store at 20 C for up to 9 months Thawed aliquots can be stored at 2 8 C for up to 6 weeks Do not refreeze the aliquots after thawing Procedure C1 Purify RNA according to the standard protocol page 14 or 20 but without performing the on column DNase digestion described in steps 12 15 instead perform the optional wash with Buffer RW1 described in step 12 C2 Mix the following in a microcentrifuge tube m A lt 87 5 pl or lt 175 pl RNA eluate m A 10 lor 20 pl Buffer RDD m A 2 5 pl or 5 pl DNase I stock solution Make the volume up to A 100 pl or 200 pl with RNase free water The reaction volumes can be doubled if necessary to A 200 pl or 40
33. iate handling of starting material b RNase contamination Use 10 mM Tris Cl pH 7 5 not RNase free water to dilute the sample before measuring purity see Appendix B page 32 For optimal results ensure that tissue samples are properly stabilized and stored in RNAlater RNA Stabilization Reagent for details see the RNAlater Handbook For frozen tissue samples ensure that they were flash frozen immediately in liquid nitrogen and properly stored at 70 C Perform the RNeasy procedure quickly especially the first few steps See Appendix A page 30 and Handling and storing starting material page 12 Although all RNeasy buffers have been tested and are guaranteed RNase ree RNases can be introduced during use Be certain not to introduce any RNases during the RNeasy procedure or later handling See Appendix A page 30 for general remarks on handling RNA Do not put RNA samples into a vacuum dryer that has been used in DNA preparations where RNases may have been used DNA contamination in downstream experiments No DNase treatment 28 Perform on column DNase digestion using the RNase Free DNase Set as described in the protocol Alternatively perform DNase digestion after RNA purification see Appendix C page 34 For real time two step RT PCR experiments carry out the RT step using the QuantiTect Reverse Transcription Kit which provides cDNA synthesis with integrated removal of genomic DNA contami
34. icroRNA from tissues and cells we recommend using miRNeasy Kits see ordering information page 39 8 RNeasy Fibrous Tissue Handbook 11 2006 RNeasy Fibrous Tissue RNeasy Fibrous Tissue Mini Procedure Midi Procedure Tissue Tissue Lyse and h jomogenize atid homogenize Proteinase K digest Add ethanol Proteinase K digest Bind total RNA Total RNA Add ethanol t tw Wash and DNase digest Bind total RNA Total RNA E Elute tom Total RNA Wash and DNase digest t E Elute Total RNA RNeasy Fibrous Tissue Handbook 11 2006 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 MH 14 3 M mercaptoethanol B ME commercially available solutions are usually 14 3 M or as alternative dithiothreitol DTT Ethanol 96 100 Sterile RNase free pipet tips Disposable gloves Water bath or heating block capable of reaching 55 C Equipment for tissue disruption and homogenization see page 12 we recommend either the TissueRuptor with TissueRuptor Disposable Probes or the Tissuelyser system see ordering information page 36 E For stabilization of RNA in tissues see page 12 RNAlater RNA Stabilization Reagent see ordering information page 36 or liquid nitrogen and
35. ll react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO When preparing Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100 C for 15 minutes Note RNeasy buffers are guaranteed RNase free without using DEPC treatment and are therefore free of any DEPC contamination 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 t Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check the supplier s instructions RNeasy Fibrous Tissue Handbook 11 2006 31 Appendix B Storage Quantification and Determination of Quality of RNA Storage of RNA Purified RNA may be stored at 20 C or 70 C in RNase free water Under these conditions no degradation of RNA is detectable afte
36. mogenized in step 4 Frozen tissues should not be allowed to thaw during handling The relevant procedures should be carried out as quickly as possible Disrupt the tissue and homogenize the lysate using either the TissueRupter follow step 4a or Tissuelyser follow step 4b See Disrupting and homogenizing starting material page 12 for more details on disruption and homogenization Note Ensure that B ME is added to Buffer RLT before use see Things to do before starting Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column Homogenization with the TissueRupter or TissueLyser generally results in higher RNA yields than with other methods Disruption and homogenization using the TissueRuptor m Place the tissue in a suitably sized vessel Add 300 pl Buffer RLT Generally round bottomed tubes allow more efficient disruption and homogenization than conical bottomed tubes m Place the tip of the disposable probe into the vessel and operate the TissueRuptor at full speed until the lysate is uniformly homogeneous usually 20 40 s Proceed to step 5 Note To avoid damage to the TissueRuptor and disposable probe during operation make sure the tip of the probe remains submerged in the buffer Disruption and homogenization using the TissueLyser m Place the tissues in the tubes prepared in step 2 m Ifthe tubes were stored on dry ice place them at room
37. nation For ordering information see page 38 RNeasy Fibrous Tissue Handbook 11 2006 Comments and suggestions RNA does not perform well in downstream experiments a Salt carryover during elution Ensure that Buffer RPE is at 20 30 C b Ethanol carryover During the second wash with Buffer RPE step 17 page 18 or 24 be sure to dry the RNeasy spin column membrane by centrifuging at 28000 x g 210 000 rpm for 2 min at 20 25 C RNeasy Fibrous Tissue Mini Kit or at 3000 5000 x g for 5 min at 20 25 C RNeasy Fibrous Tissue Midi Kit After centrifugation carefully remove the column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur To eliminate any chance of possible ethanol carryover with the RNeasy Fibrous Tissue Mini Kit place the RNeasy Mini spin column in a new 2 ml collection tube and perform the optional 1 min centrifugation step as described in step 18 of the protocol page 18 RNeasy Fibrous Tissue Handbook 11 2006 29 Appendix A General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNase
38. ng starting homogenization material page 12 for details on disruption and homogenization methods Increase g force and centrifugation time if necessary In subsequent preparations reduce the amount of starting material see page 11 and protocol page 14 or 20 and or increase the homogenization time b Too much starting material In subsequent preparations reduce the amount of starting material It is essential to use the correct amount of starting material see page 11 and protocol page 14 or 20 c Centrifugation temperature The centrifugation temperature should be too low 20 25 C Some centrifuges may cool to below 20 C even when set at 20 C This can cause formation of precipitates that can clog the RNeasy spin column If this happens set the centrifugation temperature to 25 C Warm the ethanol containing lysate to 37 C before transferring it to the RNeasy spin column Low RNA yield a Insufficient disruption and See Disrupting and homogenizing starting homogenization material page 12 for details on disruption and homogenization methods Increase g force and centrifugation time if necessary 26 RNeasy Fibrous Tissue Handbook 11 2006 Comments and suggestions b Too much starting material c RNA still bound to RNeasy spin column membrane d Ethanol carryover Low or no recovery of RNA RNase free water incorrectly dispensed RNeasy Fibrous Tissue Handbook 11 2006 In subsequent prepa
39. on an extinction coefficient calculated for RNA at neutral pH see Spectrophotometric quantification of RNA page 32 DNA contamination No currently available purification method can guarantee that RNA is completely free of DNA even when it is not visible on an agarose gel While RNeasy Kits will remove the vast majority of cellular DNA trace amounts may still remain depending on the amount and nature of the sample For analysis of very low abundance targets any interference by residual DNA contamination can be detected by performing real time RT PCR control experiments in which no reverse transcriptase is added prior to the PCR step Wilfinger W W Mackey M and Chomezynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity BioTechniques 22 474 t Values up to 2 3 are routinely obtained for pure RNA in 10 mM Tris Cl pH 7 5 with some spectrophotometers RNeasy Fibrous Tissue Handbook 11 2006 33 To prevent any interference by DNA in realtime RT PCR applications such as with ABI PRISM and LightCycler instruments we recommend designing primers that anneal at intron splice junctions so that genomic DNA will not be amplified QuantiTect Primer Assays from QIAGEN are designed for SYBR Green based real time RT PCR analysis of RNA sequences without detection of genomic DNA where possible see www qiagen com GeneGlobe For real time RT PCR assays where amplificati
40. on of genomic DNA cannot be avoided we recommend using the QuantiTect Reverse Transcription Kit for reverse transcription The kit integrates fast cDNA synthesis with rapid removal of genomic DNA contamination see ordering information page 38 Integrity of RNA The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using an Agilent 2100 bioanalyzer The respective ribosomal RNAs should appear as sharp bands or peaks The apparent ratio of 28S rRNA to 18S RNA should be approximately 2 1 If the ribosomal bands or peaks of a specific sample are not sharp but appear as a smear towards smaller sized RNAs it is likely that the sample suffered major degradation either before or during RNA purification Appendix C DNase Digestion of RNA Eluates As an alternative to on column DNase digestion steps 12 15 page 17 or 23 DNase digestion of RNA eluates can be performed instead For samples with high DNA content we recommend DNase digestion of RNA eluates as it is more efficient than on column DNase digestion Important points before starting E Donot vortex the reconstituted DNase I DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube M Inthe procedure below A refers to use of the RNeasy Fibrous Tissue Mini Kit and refers to use of the RNeasy Fibrous Tissue Mid
41. on of RNA Eluates 34 Ordering Information 36 QIAGEN Distributors and Importers 43 RNeasy Fibrous Tissue Handbook 11 2006 3 Kit Contents RNeasy Fibrous Tissue Kit Mini 50 Midi 10 Catalog no 74704 75742 Number of preps 50 10 RNeasy Mini Spin Columns each in a 2 ml Collection Tube 50 RNeasy Midi Spin Columns each in a 15 ml Collection Tube 10 Collection Tubes 1 5 ml 50 Collection Tubes 2 ml 50 Collection Tubes 15 ml 10 Proteinase K 1 4 ml 1 4 ml Buffer RLT 45 ml 45 ml Buffer RW1 45 ml 45 ml Buffer RPE concentrate 11 ml 11ml RNase Free Water 4xlOml 5x10ml RNase Free DNase Set E RNase Free DNase lyophilized 1500 units 1500 units E Buffer RDD 2x2ml 2x2ml M RNase Free Water 1 5 ml 1 5 ml Handbook 1 information working solution Shipping and Storage RNeasy Fibrous Tissue Kits are shipped at ambient temperature The RNase Free DNase Set box containing RNase free DNase Buffer RDD and RNase free water should be stored immediately upon receipt at 2 8 C The remaining components of the RNeasy Fibrous Tissue Kit should be stored dry at room temperature 15 25 C All components are stable for at least 9 months under these conditions RNeasy Fibrous Tissue Handbook 11 2006 Contains a guanidine salt Not compatible with disinfectants containing bleach See page 6 for safety Before using for the first time add 4 volumes of ethanol 6 100 as indicated on the bottle to ob
42. ore information consult the appropriate material safety data sheets MSDSs available from the product supplier 30 RNeasy Fibrous Tissue Handbook 11 2006 Glassware Glassware should be treated before use to ensure that it is RNase free Glassware used for RNA work should be cleaned with a detergent thoroughly rinsed and oven baked at 240 C for at least 4 hours overnight if more convenient before use Autoclaving alone will not fully inactivate many RNases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Fill glassware with 0 1 DEPC 0 1 in water allow to stand overnight 12 hours at 37 C and then autoclave or heat to 100 C for 15 minutes to eliminate residual DEPC Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry Solutions Solutions water and other solutions should be treated with 0 1 DEPC DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 1 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC wi
43. ormation Unless otherwise indicated perform all steps of the procedure at room temperature During the procedure work quickly Perform all centrifugation steps at 20 25 C Ensure that the centrifuge does not cool below 20 C Things to do before starting B Mercaptoethanol B ME must be added to Buffer RLT before use Add 10 pl B ME per 1 ml Buffer RLT Dispense in a fume hood and wear appropriate protective clothing Buffer RLT containing B ME can be stored at room temperature for up to 1 month Alternatively add 20 pl of 2 M dithiothreitol DTT per 1 ml Buffer RLT The stock solution of 2 M DTT in water should be prepared fresh or frozen in single use aliquots Buffer RLT containing DTT can be stored at room temperature for up to 1 month Buffer RPE is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the lyophilized DNase 1500 Kunitz units in 550 pl of the RNase free water provided in the RNase Free DNase Set box To avoid loss of DNase do not open the vial Inject RNase free water into the vial using an RNase free needle and syringe Mix gently by inverting the vial Do not vortex For long term storage of DNase remove the stock solution from the glass vial divide it into single use aliquots and store at 20 C for up to 9
44. procedure at 15 25 C Work quickly M Perform centrifugation at 20 25 C E Redissolve any precipitate in Buffer RLT by warming Add B ME before use E Before using Buffer RPE for the first time ensure ethanol is added M Prepare DNase stock solution Procedure 1 Heat water bath or heating block to 55 C 2 For the TissueRuptor disrupt and homogenize lt 250 mg tissue in 2 ml Buffer RLT For the TissueLyser disrupt and homogenize lt 150 mg tissue in 1 ml Buffer RLT and adjust volume to 2 ml with Buffer RLT 3 Add 4 ml RNase free water then 65 pl Proteinase K and mix 4 Incubate at 55 C for 20 min 5 Centrifuge at 3000 5000 x g for 5 min 6 Transfer supernatant to new tube add 0 5 volumes of 96 100 ethanol and mix Do not centrifuge 7 Transfer sample to RNeasy column in 15 ml tube Close lid centrifuge for 5 min at 3000 5000 x g and discard flow through 8 Add 2 ml Buffer RW1 to RNeasy column Close lid centrifuge for 5 min at 3000 5000 x g and discard flow through 9 Mix 20 pl DNase stock solution with 140 pl Buffer RDD add to RNeasy membrane and incubate for 15 min at 20 30 C 10 Add 2 ml Buffer RW1 to RNeasy column Close lid wait 5 min centrifuge for 5 min at 3000 5000 x g and discard flow through 11 Add 2 5 ml Buffer RPE to RNeasy column Close lid centrifuge for 2 min at 3000 5000 x g and discard flow through 12 Add 2 5 ml Buffer RPE to RNeasy column Close lid and centrifuge for 5 min a
45. r 1 year Quantification of RNA The concentration of RNA should be determined by measuring the absorbance at 260 nm Azo in a spectrophotometer see Spectrophotometric quantification of RNA below For small amounts of RNA however it may be difficult to determine amounts photometrically Small amounts of RNA can be accurately quantified using an Agilent 2100 bioanalyzer quantitative RT PCR or fluorometric quantification Spectrophotometric quantification of RNA To ensure significance Aygo readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 pg of RNA per ml Ayo 1 gt 44 pg ml This relation is valid only for measurements at a neutral pH Therefore if it is necessary to dilute the RNA sample this should be done in a buffer with neutral pH As discussed below see Purity of RNA page 33 the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity When measuring RNA samples be certain that cuvettes are RNase free especially if the RNA is to be recovered after spectrophotometry This can be accomplished by washing cuvettes with 0 1 M NaOH 1 mM EDTA followed by washing with RNase free water see Solutions page 31 Use the buffer in which the RNA is diluted to zero the spectrophotometer An example of the calculation involved in RNA quantification is shown below Volume of RNA sample 100 pl Dilution 10 pl of RNA sample 490 pl of 10 mM
46. ration Mixing should only be carried out by gently inverting the tube Do not vortex 14 Add the DNase incubation mix 160 pl directly to the RNeasy spin column membrane and place on the benchtop 20 30 C for 15 min Note Be sure to add the DNase incubation mix directly to the RNeasy spin column membrane DNase digestion will be incomplete if part of the mix sticks to the walls or the O ring of the spin column 15 Add 2 ml Buffer RW1 to the RNeasy spin column Close the lid gently and wait for 5 min and then centrifuge for 5 min at 3000 5000 x g at 20 25 C Discard the flow through v 2 wn A A am 2 oo 2 z 2 S2 wn e Z z Reuse the collection tube in step 16 16 Add 2 5 ml Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge at 20 25 C for 2 min at 3000 5000 x g to wash the membrane Discard the flow through Reuse the collection tube in step 17 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting 17 Add 2 5 ml Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge at 20 25 C for 5 min at 3000 5000 x g to wash the membrane The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the RNeasy spin column
47. rations reduce the amount of starting material see page 11 and protocol page 14 or 20 and or increase the volume of lysis buffer and the homogenization time In subsequent preparations reduce the amount of starting material It is essential to use the correct amount of starting material see page 11 and protocol page 14 or 20 Repeat RNA elution but incubate the RNeasy spin column on the benchtop for 10 min with RNase free water before centrifuging During the second wash with Buffer RPE step 17 page 18 or 24 be sure to dry the RNeasy spin column membrane by centrifuging at gt 8000 x g 210 000 rpm for 2 min at 20 25 C RNeasy Fibrous Tissue Mini Kit or at 3000 5000 x g for 5 min at 20 25 C RNeasy Fibrous Tissue Midi Kit After centrifugation carefully remove the column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur To eliminate any chance of possible ethanol carryover with the RNeasy Fibrous Tissue Mini Kit place the RNeasy Mini spin column in a new 2 ml collection tube and perform the optional 1 min centrifugation step as described in step 18 of the protocol page 18 Add RNase free water to the center of the RNeasy spin column membrane to ensure that the membrane is completely covered 27 Comments and suggestions Low Aygo Aogo Value Water used to dilute RNA for A260 A280 Measurement RNA degraded a Inappropr
48. re starting E If using RNeasy Fibrous Tissue Kits for the first time read Important Notes page 11 MH If working with RNA for the first time read Appendix A page 30 E If using the TissueRuptor ensure that you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor Handbook E If using the TissueLlyser ensure that you are familiar with operating it by referring to the operating instructions and TissuelLyser Handbook MH Since the RNase inactivating Buffer RLT must be diluted to permit proteinase K digestion this protocol should not be used for tissues rich in RNases such as pancreas or intestine E Fresh frozen or RNAlater stabilized tissues can be used If freezing tissues flash freeze in liquid nitrogen and immediately transfer to 70 C where they can be stored for several months Do not allow tissues to thaw during weighing or handling prior to disruption in Buffer RLT Homogenized tissue lysates from step 4 can also be stored at 70 C for several months Incubate frozen lysates at 37 C in a water bath until completely thawed and salts are dissolved before continuing with step 5 Avoid prolonged incubation which may compromise RNA integrity E Do not vortex reconstituted DNase I DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube E Buffer RIT may form a precipitate upon storage If necessary redissolve by
49. s prevention or treatment of a disease The RNeasy Mini Kit is for general laboratory use No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease Visit www giagen com geneXpression to find out more about standardized solutions for gene expression analysis from RNA preparation to real time RT PCR Larger kit size available see www giagen com RNA t Requires use of the Plate Rotor 2 x 96 and Centrifuge 4K15C Tissuelyser system recommended for disruption and homogenization QlAvac 96 optional RNeasy Fibrous Tissue Handbook 11 2006 39 Notes 40 RNeasy Fibrous Tissue Handbook 11 2006 Notes RNeasy Fibrous Tissue Handbook 11 2006 41 Notes 42 RNeasy Fibrous Tissue Handbook 11 2006 QIAGEN Distributors and Importers Please see the back cover for contact information for your local QIAGEN office Argentina Egypt Lithuania Singapore Tecnolab S A Clinilab INTERLUX Research Biolabs Pte Lid Tel 011 4555 0010 Tel 52 57 212 Tel 370 5 2786850 Tel 6777 5366 Fax 011 4553 3331 Fax 52 57 210 Fax 370 5 2796728 Fax 6778 5177 E mail info tecnolab com ar Email Clinilab link net E mail spirit interlux It Email sales researchbiolabs com Bosnia Herzegovina Estonia Malaysia Slovak Republic MEDILINE d o o Quantum Eesti AS RESEARCH BIOLABS SDN BHD BIO CONSUIT Slovakia spol s r o Tel 386 1 8
50. s into the RNA sample during or after the purification procedure In order to create and maintain an RNase free environment the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep purified RNA on ice when aliquots are pipetted for downstream applications Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally RNase free and do not require pretreatment to inactivate RNases Nondisposable plasticware Nondisposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 31 Alternatively chloroform resistant plasticware can be rinsed with chloroform to inactivate RNases When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For m
51. ssueRuptor 120 V 60 Hz for North America and Japan t 235 V 50 60 Hz for Europe excluding UK and Ireland 235 V 50 60 Hz for UK and Ireland 235 V 50 60 Hz for Australia 36 75742 76104 76106 76154 76163 9001271 9001272 9001273 90012748 990890 RNeasy Fibrous Tissue Handbook 11 2006 Ordering Information Product Contents Cat no Tissuelyser Universal laboratory mixer mill 85200 disruptor 852101 85220 Tissuelyser Adapter Set 2 x 24 2 sets of Adapter Plates and 2 racks 69982 for use with 2 ml microcentrifuge tubes on the TissueLyser Tissuelyser Single Bead For dispensing individual beads 69965 Dispenser 5 mm 5 mm diameter Stainless Steel Beads Stainless Steel Beads suitable for 69989 5 mm 200 use with the TissueLyser system Buffer RLT 220 ml 220 ml lysis buffer for RNeasy Kits 79216 QIAGEN Proteinase K 2 ml 2 ml gt 600 mAU nIl solution 19131 QIAGEN Proteinase K 10 ml 10 ml gt 600 mAU ml solution 19133 RNase Free DNase Set 50 1500 units RNase Free DNase I 79254 RNase Free Buffer RDD and RNase Free Water Collection Tubes 2 ml 1000 x 2 ml Collection Tubes 19201 Tissuelyser Adapter Set 2x96 2 sets of Adapter Plates for use 69984 with Collection Microtubes racked on the Tissuelyser Collection Microtubes racked Nonsterile polypropylene tubes 19560 1 2 ml 960 in racks of 96 Collection Microtube Nonsterile polypropylene caps for 19566 Caps 120 x 8
52. t 3000 5000 x g 13 Place RNeasy column in new 15 ml tube Add 150 pl RNase free water close lid wait 1 min and centrifuge for 3 min at 3000 5000 x g Optional Repeat elution with another volume of water or with RNA eluate Please tear off here
53. tain a The ready to use proteinase K included in the kit is dissolved in a specially formulated storage buffer The proteinase K is stable for up to 1 year after delivery when stored at room temperature To prolong the lifetime of the proteinase K we recommend storage at 2 8 C Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of RNeasy Fibrous Tissue Mini Kit and RNeasy Fibrous Tissue Midi Kit is tested against predetermined specifications to ensure consistent product quality Product Use Limitations RNeasy Fibrous Tissue Kits are intended for research use No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance an
54. tions Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur 18 Optional Place the RNeasy spin column in a new 2 ml collection tube supplied and discard the old collection tube with the flow through Close the lid gently and centrifuge at full speed for 1 min Perform this step to eliminate any possible carryover of Buffer RPE or if residual flow through remains on the outside of the RNeasy spin column after step 17 Flow through contains Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information 18 RNeasy Fibrous Tissue Handbook 11 2006 19 Place the RNeasy spin column in a new 1 5 ml collection tube supplied Add 30 50 pl RNase free water directly to the RNeasy spin column membrane Close the lid gently To elute the RNA centrifuge for 1 min at gt 8000 x g 210 000 rpm at 20 25 C 20 Repeat step 19 using another 30 50 pl RNase free water or using the eluate from step 19 if high RNA concentration is required Reuse the collection tube from step 19 If the expected RNA yield is gt 30 pg there is no need to repeat step 19 If using the eluate from step 19 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but the final RNA concentration will be higher A Z ts fe a x lt ay o 2 e a a a c
55. warming and then place at room temperature 15 25 C 14 RNeasy Fibrous Tissue Handbook 11 2006 Buffer RIT and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 6 for safety information Unless otherwise indicated perform all steps of the procedure at room temperature During the procedure work quickly Perform all centrifugation steps at 20 25 C Ensure that the centrifuge does not cool below 20 C Things to do before starting B Mercaptoethanol B ME must be added to Buffer RLT before use Add 10 pl B ME per 1 ml Buffer RLT Dispense in a fume hood and wear appropriate protective clothing Buffer RLT containing B ME can be stored at room temperature for up to 1 month Alternatively add 20 pl of 2 M dithiothreitol DTT per 1 ml Buffer RLT The stock solution of 2 M DTT in water should be prepared fresh or frozen in single use aliquots Buffer RLT containing DTT can be stored at room temperature for up to 1 month Buffer RPE is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the lyophilized DNase 1500 Kunitz units in 550 pl of the RNase free water provided in the RNase Free DNase Set box To avoid loss of DNase do not open the vial Inject RNase fr
56. win com cn BioMarker Kft ext 301 310 311 Taiwan Tel 36 28 419 986 Fax 971 4 269 0612 TAIGEN Bioscience Corporation Gene Company Limited Fax 36 28 422 319 ATTN LAB DIVISION Tel 02 2880 2913 Tel 86 21 64951899 Email biomarker biomarkerhy Email shaji almaz net ae Fax 02 2880 2916 Fax 86 21 64955468 E mail order taigen com Email India Pakistan info_bj genecompany com Beijing _ Genetix Pakistan Microbiological Associates Thailand info_sh genecompany com Shanghai Tel 91 11 51427031 Tel 92 51 5567953 Theera Trading Co Ltd info_cd genecompany com Chengdu Fax 91 11 25419631 Fax 92 51 5514134 Tel 02 412 5672 info_gz genecompany com E mail genetix genetixbiotech com Email orderpma comsats net pk Fax 02 412 3244 Guangzhou E mail theetrad samart co th Indonesia Peru Genetimes Technology Inc PT Research Biolabs Turkey Order 3008200465 Tel 62 21 5865357 INMUNOCHEM SAC Medek Medikal Ur nler F Tel 51 1 4409678 k 2 Tel 86 21 54262677 E mail ve Saglik Hizmetleri A S i 3 A Fax 51 1 4223701 Fax 86 21 64398855 indonesia researchbiolabs com Endi anmiunischep teia oe Tel 216 302 15 80 Email order genetimes com cn l g JRPS iFa 216 302 15 88 ron E mail makialp med ek com Colombia Zist Baran BIORAIN Poland z Tel 98 21 88066348 or Syngen Biotech Sp z 0 0 United Arab Emirates SENTECH Conefics amp Tecnology 98 21 88066349 Tek 071 798 5850 52 Al Mazouri Medical amp Chemi

RNeasy Fibrous Tissue Handbook

Contents

Download Pdf Manuals

Related Search

Related Contents