FastRNA® Pro Soil
Contents
1. the tube side and it may appear that RNA has not been purified Complete the protocol and confirm the RNA concentration by and integrity by gel electrophoresis RNA adhering to the tube wall will not affect its purity size or use in subsequent applications Following RNA precipitation the RNA pellet may not be firmly attached to the side or bottom of the tube and may be observed floating in the solution or at the solution surface Re centrifuge the sample in the same tube and exercise caution to not lose the pellet when removing the supernatant A brown color present in the RNA pellet after Step 21 is most likely due to co purification of humic substances which will be removed by the RNAMATRIX in steps 25 33 It has been determined that in some instances RNA dilution 1 3 1 5 or 1 10 may result in greater yield of PCR product It is important to recognize that some soil samples may not RT PCR amplify due to purification of small amounts of total or target RNA that result from a low organism content or the soil sample may be exceptionally high in inhibiting substances including nonspecific humic substances If dilution of the RNA sample and nested or reamplification of the PCR reaction do not facilitate successful RI PCR it is recommended the samples receive additional purification using the kit provided Quick Clean Spin Filters 8 4 Genomic DNA Contamination The FastRNA Pro Soil Indirect Kit is designed to remove genomic2. the stability samples over extended time periods eg hours days weeks to provide information about the relative RNA yields and losses that can be expected during storage Aliquots of the control microorganism may also be stored without soil and processed in parallel to compare RNA yield with the soil stability samples Lack of RNA degradation in the non soil contro tube indicates the soil stability sample RNA was likely degraded during soil storage prior to the addition of RNApro Solution Certain bacterial strains may contain elevated RNase levels Reduce the exposure time to RNase by adding the RNApro Solution to each sample as soon as possible following sample harvest RNApro Solution will protect RNA in soil samples from degradation for at least 24 hours at room temperature or 4 C 8 2 Suspected RNA Degradation The quality of RNA can be determined after electrophoresis by the appearance of distinct large and small ribosomal RNAs rRNA of approximately 0 9 to 1 5 kb Due to the potential organism heterogeneity in a soil sample multiple bands may be present The purified rRNA concentration may appear low but is not completely indicative of the amount of mRNA present in the sample RT PCR will often yield positive results in the absence of visible rRNA MessengerRNA mRNA whichtypically represents approximately 1 of the total cellular RNA and is heterogeneous length may not be highly visible Ribosomal RNA is used as a m
3. 0 9 to 1 5 kb Due to the potential organism heterogeneity in a soil sample multiple bands may be present The purified rRNA concentration may appear low but is not completely indicative of the amount of mRNA present in the sample RT PCR will often yield positive results in the absence of visible rRNA Evaluate the purified RNA for use in RT PCR If the purified RNA appears colorless it is acceptable for use in Northern analysis and should perform satisfactorily in RT PCR For RT PCR amplification it is recommended to test ul undiluted and ul of RNA diluted 1 3 1 5 and 1 10 If the RNA does not amplify satisfactorily continue with additional purification using Quick Clean Spin Filters to remove residual inhibiting substances Section 6 Aliquot and store the RNA at 70 C NOTE RNA is generally stable for up to a year at 70 to 80 C For longer term storage RNA samples may be stored as ethanol precipitates When stored as an ethanol precipitate the RNA must be spun down washed and resuspended in aqueous solution prior to use Avoid frequent sample freeze thaw by storing isolated RNA as aliquots NOTE RNA does not evenly distribute in ethanol and can lead to inconsistent RNA amounts between samples when equal volumes are pipetted Vortex the RNA ethanol solution to disperse the RNA prior to removing the sample In situations where precise amounts of RNA are required it is best to precipitate the total amount or exce
4. Applications The FastRNA Pro Soil Indirect Kit selectively purifies total cellular RNA free from DNA protein and soil components that is sufficiently pure for use in RT PCR and Northern analysis While quality control tests indicate DNA removal during RNA purification the user may incorporate DNase treatment of the RNA prior to use in applications where absolute control of DNA contamination is required Use DNase at the concentration and incubation conditions recommended by the manufacturer DNase is inactivated by incubation at 75 C for 5 minutes or by addition of EDTA to 25 mM followed by phenol chloroform extraction and precipitation 13 14 FastRNA Pro Soil Indire The FastRNA Pro Soil Indirect Kit is designed to provide two levels of RNA purification The first level the basic protocol in Section 5 incorporates the proprietary RNAMATRIX to remove soil associated reverse transcription and PCR inhibitors to allow amplification of undiluted RNA for the vast majority of soil types It has been determined that in some instances RNA dilution 1 3 1 5 or 1 10 of clear or slightly colored samples may result in greater yield of PCR product It is important to recognize that some soil samples may not RT PCR amplify due to purification of small amounts of total or target RNA that result from a low organism content or the soil sample may be exceptionally high in inhibiting substances including nonspecific humic subst
5. DNA during sample processing However if genomic DNA contamination is suspected it will appear as a high molecular weight smear on a denaturing gel Genomic DNA contamination and or protein contamination may appear during agarose electrophoresis as ethidium bromide stained material in the gel loading well To remove the DNA and or protein re extract the RNA sample with phenol pH 52 saturated with 0 1 M Tris chloroform or chloroform isoamyl alcohol 24 1 The lower phase of the extraction contains the genomic DNA protein will accumulate at the organicaqueous interface Both the lower phase and the interphase protein should be carefully avoided when removing the top aqueous RNA containing phase Leaving a small volume of the top phase in the tube will help prevent accidental DNA or protein contamination 8 5 RT PCR Inhibition The FastRNA Pro Soil Indirect Kit is designed to provide levels of RNA purification that permit tailoring the protocol to the soil sample and the amount of reverse transcription and PCR inhibitors present in the soil sample Steps 33 provide the first level of purification using proprietary RNAMATRIX Section 6 23 FastRNA Pro Soil Indi 24 provides second level purification through Ouick Clean Spin Filters RNAMATRIX purification provides sufficient RNA purification for the majority of soil samples that permits RNA use in Northern analysis and RT PCR amplification without additional purific
6. ances If dilution of the RNA sample and nested or reamplification of the PCR reaction do not facilitate successful RT PCR the samples can be additionally purified using the Quick Clean Spin Filters provided in the kit Section 6 Centrifugation of purified RNA through the Quick Clean Spin Filters as directed will remove residual inhibitors with no significant loss of RNA quantity 4 Safety Precautions RNApro Solution contains components that when in contact with human tissue or during inhalation may cause irritation or burning Wear personal protective equipment to prevent skin contact e g gloves lab coat and eye protection prevent inhalation of reagent vapors and consumption of liquid during use and dispose of waste following proper procedures Consult the enclosed Material Safety Data Sheet for additional details 5 Basic Protocol for All Soil Samples Transfer gram of soil to a 15 ml conical tube NOTE Greater than g of soil may be used if the soil is known to contain few microorganisms or if the RNA yield is expected to be low Also wet soils often provide an enriched growth environment that results in greater organism diversity and RINA yield but since a sizeable proportion of the sample weight is due to water content more soil may be needed Finally numbers of viable soil microorganisms can be increased prior to RINA isolation by incubating the soil in a liquid growth media and then proceeding directly to Ste
7. arker to assess sample degradation Degraded RNA or mRNA may appear as 21 FastRNA Pro Soil Indi 22 unequal fluorescent intensity between bands a single rRNA band may be completely lacking or a heterogeneous fluorescent smear may appear below the rRNA bands The rRNA content is not an accurate indication of mRNA content purified from soil Samples that lack visible rRNA in agarose gel electrophoresis will often function successfully in RT PCR amplification RNase may have been introduced during isolation To prevent RNAse contamination the use of gloves RNase free plugged pipette tips and RNase free tubes is strongly recommended Clean pipetmen and work area with RNase Erase Catalog 2440 204 prior to beginning RNA isolation Use DEPC treated reagents RNApro Solution contains RNase inactivating components and will not support RNase contamination Ensure that DEPC treated H O was used to make the 7076 ethanol Artifactual RNA degradation may occasionally occur during gel electrophoresis due to a gel that was not RNase free running the gel at too high voltage or from using depleted running buffer Rerun the samples with a known intact RNA sample using freshly prepared RNase free electrophoresis reagents 8 3 Properties of the RNA Pellet Following RNA precipitation the purified RNA may not appear as a pellet at the tube bottom but may instead adhere to the side of the tube The RNA may not be visible in the pellet or on
8. ation It has been demonstrated that in some instances RNA dilution prior to amplification 1 3 1 5 or 1 10 may result in greater yield of PCR product It is important to recognize that individual soil samples that are resistant to RT PCR may have a low amount of RNA due to low organism content or may be so high in inhibiting substances including nonspecific humic acids that dilution of the RNA sample still does not allow successful RT PCR For these samples MP Biomedicals has included Quick Clean Spin Filters in the FastRNA Pro Soil Indirect Kit as an optional last step Centrifugation of purified RNA through the Quick Clean Spin Filter as directed will remove any residual enzymatic inhibitors with no significant loss of RNA quantity Unsuccessful RT PCR may also result from the inadvertent introduction of RNase into RT PCR reagents during experimental handling Include a control RNA with the RT PCR reagents to test for RNA degradation Unsuccessful RT PCR may result if the reverse transcriptase and or the thermostable polymerase is inactive or was not added to the reaction or if other solutions are compromised or omitted Perform RT PCR using enzymes and buffers with a known control RNA and primers Unsuccessful RT PCR may also result if PCR primer conditions have not been optimized Test the amplification primers using a control RNA to confirm the ideal annealing temperature and concentration 8 6 Mucopolysaccharide Carbohy
9. centration and integrity a Dilute 5 ul of the purified RNA into 495 ul of DEPC H O b Read the OD using DEPC H2O as a blank c Calculate the sample ug RNA per ml using the formula OD 40 ug ml per OD 100 dilution factor ug RNA per ml Spectrophotometer accuracy is greatest between 0 2 and 0 8 If the OD reading is below this range add more RNA sample e g 20 ul RNA 480 ul DEPC H O or concentrate the RNA by precipitation and resuspension into a smaller volume If the OD reading is above the recommended spectrophotometer range use less RNA for the OD determination If RNA yield is low greater accuracy will be achieved by concentrating the RNA sample before analysis or use agarose gel electrophoresis to approximate the concentration FastRNA Pro Soil Indii 35 36 RNA integrity and an estimation of yield can be determined by analyzing a portion of the RNA sample using gel electrophoresis and comparing it to a known amount of RNA Take a 15 ul aliquot of RNA add gel loading buffer and load the sample and the known amount of RNA on a 1 0 agarose gel at 100 volts for 30 minutes Ethidium bromide may be added to the denatured RNA sample at 10 ug per milliliter prior to gel loading or the gel may be ethidium bromide stained and destained following electrophoresis and visualized under UV light The quality of the RNA is determined by the appearance of distinct large and small ribosomal RNAs of approximately
10. drate Contamination Cellular mucopolysaccharides will not co purify with RNA using the protocol and reagents in the FastRNA Pro Soil Indirect Kit 9 Recommended Reference Format for Publication Total RNA was isolated from g type soil using the FastRNA Pro Soil Indirect Kit MP Biomedicals Irvine CA and FastPrep 24 Instrument MP Biomedicals Irvine CA Samples have been homogenized for seconds at a speed setting of 10 References Faegri A V L Torsvik and J Goks yr 1977 Bacterial and fungal activities in soil separation of bacteria and fungi by a rapid fractionated centrifugation technique Soil Biol Biochem 9 105 112 2 Roszak D B and R R Colwell 1987 Survival strategies of bacteria in the natural environment Microbiol Rev 51 365 379 3 Selenska S and W Klingm ller 1991 DNA recovery and molecular analysis of DNA and RNA from soil Microb Releases 41 46 4 Bej A K M Perlin and R M Atlas 1991 Effect of introducing genetically engineered microorganisms on soil microbial community diversity FEMS Microbiol Ecol 86 169 175 5 Ka JO Z Yu and W W Mohn 2001 Monitoring the Size and Metabolic Activity of the Bacterial Community during Biostimulation of Fuel Contaminated Soil using Competitive PCR and RT PCR Microb Ecol 42 267 273 6 Hurt RA X Qiu L Wu Y Roh A V Palumbo J M Tiedje and J Zhou 2001 Simultaneous recovery of RNA and DNA from soi
11. e Application Manual Rapid Isolation of Total RNA from Soil Using the FastPrep and FastPrep 24 Instruments Catalog 6075 050 50 Preps Storage Ambient temperature 15 30 C DO NOT expose Phenol Chloroform 1 1 to light for extended periods of time Store in the original amber bottle in the closed kit box Revision 6075 050 06APR MP Biomedicals e 29525 Fountain Parkway e Solon OH 44139 e tel 1 800 854 0530 fax 1 800 334 6999 FastRNA Pro Soil Indirect Kit FastRNA Pro Soil Indirect Kit Rapid Isolation of Total RNA from Soil Using the FastPrep and FastPrep 24 Instruments Catalog 6075 050 50 Preps Storage Ambient temperature 15 30 C DO NOT expose Phenol Chloroform 1 1 to light for extended periods of time Store in the original amber bottle in the closed kit box Revision 6075 050 06APR TABLE OF CONTENTS LA 9 10 References Related Introduction to the FastRNA Pro Soil Indirect Kit and the FastPrep Instruments saat Ste Kit Components and 7 Supplied Materials is O 2 FastRNA Pro Soil Indirect Kit GE el 6 2 2 User Supplied Materials senn 7 Important Considerations before 7 3 1 Preparation of RNAMATRIX Wash Schon 3 2 Separation of Organic Materials from Soil 8 3 3 Preparing to isolate total RINA 34 Sample Lysis with the FastPrep In
12. e total RNA isolated from four different soil samples was loaded on to a 0 8 agarose gel Lane Ikb ladder Lane 2 RNA from 5 g Soil 1 Lane 3 RNA from 10 g Soil 2 Lane 4 RNA from 5 g Soil 7 Lane 5 RNA from 5 g Soil 10 FastRNA Pro 20 PICTURE 2 FastRNA Pro Soil Indirect Kit RT PCR of Fungal Gene from Total RNA Isolated from Soil Samples with the FastRNA Pro Soil Indirect Kit Approximately 40 of the RT PCR reaction was loaded on to a 0 8 agarose gel Lane 1506 2kb marker Lane 2 Soil 1 Lane 3 Soil 2 Lane 4 Soil 7 Lane 5 Soil 10 8 Troubleshooting 8 1 Lower Than Expected or No RNA Yield Due to natural soil diversity soil samples may contain very low amounts of the desired organism s for extracting RNA Additional numbers of the same sample may be processed using multiple tubes and the purified RNA pooled Alternatively a greater amount of soil can be used in step of the protocol in section 5 Soil samples stored for extended periods may result in organism and RNA deterioration To prevent sample deterioration process the sample immediately following collection In order to understand storage deterioration in specific soil types a control stability experiment using a laboratory microorganism i e E coli or S cereviseae stored in the soil sample may be performed Add equivalent amounts of microorganism to aliquots of the soil and store using the standard method Prepare RNA from
13. estion sonication blending douncing and vortexing Soil biodiversity is directly affected by its physical and chemical composition and by environmental factors Evidence indicates total soil biodiversity can be underestimated by approximately 90 when an in vitro culturing method is used to approximate the total number of organisms present 1 2 For this reason extraction of total RNA from soil has been used to detect specific genes from unculturable microorganisms to provide a method to isolate and identify individual strains of interest estimate soil biodiversity estimate soil microorganism metabolic activity and to clone expressed genes 3 4 5 6 Nucleic acid extraction from soil can be performed using a direct or an indirect method The indirect method utilizes an initial separation of microorganisms and other biological specimens from the soil followed by lysis of the organisms and RNA purification 7 8 The indirect method also permits soil incubation with growth media to amplify living organisms prior to RNA isolation if accurate measurements of microbial diversity are not required The direct method consists of extracting nucleic acid from microorganisms and other biological specimens directly from soil 9 FastRNA Pro Soil Kits are available from MP Biomedicals for both direct Cat 6070 050 and indirect 6075 050 RNA isolation methods FastRNA Pro Soil Indi Soil types differ in the type and amount of organic
14. for 5 minutes Microcentrifuge pulse spin approximately 0 seconds to pellet the RNAMATRIX bound RNA and discard the supernatant Use caution not to remove the RNAMATRIX Completely resuspend the RNAMATRIX bound RNA in 500 ul of prepared RNAMATRIX Wash Solution NOTE Ensure that 15 ml of ethanol has been added to the RNAMATRIX Wash Solution Concentrate prior to use Microcentrifuge pulse spin approximately 0 seconds and discard the supernatant Use caution not to remove the RNAMATRIX Microcentrifuge pulse spin a second time for approximately IO seconds and carefully remove any residual wash solution with a pipet Use caution not to remove the RNAMATRIX 30 3l 32 33 34 Air dry 5 minutes at room temperature Add 50 ul of DEPC H O and completely resuspend the RNAMATRIX by vortexing Incubate 5 minutes at room temperature to elute the Finger tap the tube bottom frequently to provide gentle mixing Microcentrifuge pulse spin approximately 10 seconds and transfer the supernatant containing eluted RNA to a new tube NOTE Do not discard the RNAMATRIX pellet Repeat step 3l and 32 to provide a final RNA volume of 100 ul NOTE If matrix carryover occurs remove the matrix by pulse centrifuging the microcentrifuge tube for approximately 10 seconds to pellet the matrix Carefully transfer only the supernatant to a new RNase free microcentrifuge tube Determine the RNA con
15. igh in inhibiting substances including nonspecific humic substances If dilution of the RNA sample and nested or reamplification of the PCR reaction do not facilitate successful RT PCR the samples can be additionally purified using the Quick FastRNA Pro Soil Indirect Clean Spin Filters Centrifugation of purified RNA through the Quick Clean Spin Filter as directed will remove residual inhibitors with no significant loss of RNA quantity If the RNA sample is frozen thaw completely and centrifuge briefly to bring the liquid to the tube bottom before proceeding Applying 50 DEPC H O to the Quick Clean Spin Filter insert the filter into a usersupplied RNase free microcentrifuge tube and pulse spin for 10 seconds Transfer the Quick Clean Spin Filter to a new kit supplied RNase free Catch Tube insert into the microcentrifuge rotor and apply the RNA up to 300 ul may be processed to the Quick Clean Spin Filter NOTE Do not leave the RNA in contact with the Quick Clean Spin Filter for more than 60 seconds before pulse spinning for 10 seconds in the next step or RNA loss will occur Pulse spin the Quick Clean Spin Filter and Catch Tube for 10 seconds to collect purified RNA Quantify the RNA per Step 34 in section 5 7 Example Data Total RNA Isolation and RT PCR PICTURE 1 FastRNA Pro Soil Indirect Kit Total RNA extracted from Soil Samples with the FastRNA Pro Soil Indirect Kit Approximately 15 of th
16. l Inhibitor Removal Solution 12 ml RNAMATRIX Slurry 0 6 ml RNAMATRIX Binding Solution 35 ml RNAMATRIX Wash Solution Concentrate 15 ml DEPC Treated H O 30 ml Lysing Matrix E 50 tubes Ouick Clean Spin Filters 50 filters Catch Tubes 50 tubes User Manual each MSDS each Certificate of Analysis each 2 2 User Supplied Materials Resuspension Solution either deionized H2O 10 mM Tris pH 7 0 8 0 or 100 mM NaCl Cheesecloth VWR Grade 10 RNase Erase See Section Related Products FastPrep or FastPrep 24 Instrument See Section Related Products 100 Ethanol Chloroform Centrifuge capable of spinning at least 10 ml of liquid at less than 500 rpm 20 x g Centrifuge capable of spinning at least 10 ml of liquid at 5000 rpm 1 800 x g Microcentrifuge Chilled 70 Ethanol prepared with DEPC treated H O Chilled Isopropanol 1 5 or 2 0 ml RNase free Microcentrifuge Tubes Agarose Gel Loading Dye Size Marker for Electrophoresis 3 Important Considerations before Use 3 1 Preparation of RNAMATRIX Wash Solution The FastRNA Pro Soil Indirect Kit contains a bottle with 15 ml of RNAMATRIX Wash Solution Concentrate Before using this solution add an equal volume 15 ml of 100 ethanol and mark on the bottle label the date ethanol was added Store at room temperature FastRNA Pro Soil Indit 3 2 Separation of Organic Material from Soil The FastRNA Pro Soil Indirect Kit protocol i
17. l by pipetting or vortexing Transfer ml of the resuspended material to a Lysing Matrix E tube provided in the kit Securely close the cap to prevent leakage in the next step NOTE The designated volumes ensure adequate airspace in the matrix tube to prevent sample leakage and or tube failure DO NOT overfill the matrix tube Use a second matrix tube to process a larger sample Process the sample in the FastPrep or FastPrep 24 Instrument for 40 seconds at a setting of 6 0 Remove the tube and centrifuge at 2 14 000 x g for 5 minutes at room temperature Transfer the liquid to a microcentrifuge tube NOTE Minimal debris carryover will not affect subsequent steps Incubate the sample 5 minutes at room temperature to increase RNA yield Add 300 ul of usersupplied chloroform NO isoamyl alcohol and vortex 10 seconds 20 21 Incubate 5 minutes at room temperature to permit nucleoprotein dissociation and increase RNA purity Centrifuge at 2 14 000 x g for 5 minutes at 4 C Remove the upper aqueous phase to a new microcentrifuge tube without disturbing the interphase NOTE Ifa portion ofthe interphase is accidentally transferred repeat the centrifugation in step 15 with the contaminated upper phase and transfer the new upper phase to a clean microcentrifuge tube Add 200 yl of Inhibitor Removal Solution Invert 5 times to completely mix Centrifuge at gt 4000 x g for 5 mi
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19. ls and sediments Appl Environ Microbiol 67 4495 503 25 FastRNA Pro Soil Indire 26 Ogram A G 5 Sayler and T Barkay 1987 The extraction and purification of microbial DNA from sediments J Microb Methods 7 57 66 Torsvik V L 1980 Isolation of bacterial DNA from soil Soil Biol Biochem 12 15 21 Holben W E J K Jansson B K Chelm and J M Tiedje 1988 DNA probe method for the detection of specific microorganisms in the soil bacterial community Appl Environ Microbiol 54 703 7 1 1 Humus Chemistry Stevenson FJ John Wiley amp sons Inc 2nd Edition 1994 Tebbe C C and Wilfried Vahjen 1993 Interference of Humic Acids and DNA Extracted Directly from Soil in Detection and Transformation of Recombinant DNA from Bacteria and Yeast Appl Environ Microbiol 59 2657 2665 Tsai Y L and B H Olson 1992 Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction Appl Environ Microbiol 58 754 757 Molecular Cloning Sambrook and Russel Cold Spring Harbor Laboratory Press 3rd Edition 2001 Current Protocols in Molecular Biology John Wiley amp Sons Inc 2002 www currentprotocols com 11 Related Products 11 1 FastRNA Pro Soil Direct Kit Cat 6070 050 The FastRNA Pro Soil Direct Kit is designed to efficiently isolate total RNA from organic material found in soil samples Cellular material is washed away f
20. materials The largest and most chemically significant fraction of natural organic matter is the humic substances which include humic acid and fulvic acid 10 The amount and type of humic substances in a soil sample are established by a combination of environmental conditions vegetation and topography and will vary among soil types and even within soil at the same location Humic substances frequently give soil a yellow brown color and have been shown to inhibit Taq polymerase activity at concentrations as low as 0 l ug ml 11 12 The FastRNA Pro Soil Kits purify RNA in a process that removes humic substances and other inhibitors and efficiently inactivates cellular RNases during homogenization to prevent RNA degradation The FastRNA Pro Soil Indirect Kit offers two levels of RNA purification that permit tailoring the protocol to the soil sample and downstream applications In the first level RNA is purified from contaminating soil products by selective binding to RNAMATRIX For the vast majority of soil types RNAMATRIX purification will provide RNA that is colorless and free of RI PCR inhibitors for use in downstream applications In the event further processing is required a second level of purification through Quick Clean Spin Filters will provide additional purification of colorless and contaminant free RNA 2 Kit Components and User Supplied Materials 2 1 FastRNA Pro Soil Indirect Kit Components RNApro MSolution 55 m
21. ncorporates incubating the soil sample with Resuspension Solution to separate microorganisms and other biological specimens from the soil using low speed centrigation prior to lysis and RNA purification The supernatant is then filtered through cheesecloth to remove remaining soil particles NOTE It is important NOT to substitute sterile gauze Kim Wipe tissue or commercially available coffee filters as significant RNA yield reduction will occur Following filtration microorganisms and biological specimens are concentrated from the Resuspension Solution by a second centrifugation Pelleted organic material is then lysed in RNApro Solution by the FastPrep or FastPrep 24 Instrument 3 3 Preparing to Isolate Total RNA The presence or introduction of RNase during the procedure may result in sample degradation It is strongly recommended that the user minimize the potential for RNase contamination by wearing gloves throughout the procedure using DEPC H O and by treating pipettmen work area gel box and gel comb with RNase Erase see section 2 2 Additional RNA handling methods and precautions are found in references 13 and 4 Confirm the Lysing Matrix E tubes spin freely and will not scrape the microcentrifuge wall during centrifugation Add RNApro Solution to the sample as soon as possible to initiate RNase inhibition FastPrep Instrument homogenized and non homogenized samples are stable in RNApro Solution for up to 24 h
22. ntal clinical investigation These products are not to be used as food food additives or general household items Purchase of MP Biomedicals products does not grant rights to reproduce modify or repackage the products or any derivative thereof to third parties MP Biomedicals makes no warranty of any kind expressed or implied including merchantability or fitness for any particular purpose except that the products sold will meet our specifications at the time of delivery Buyer s exclusive remedy and the sole liability of MP Biomedicals hereunder shall be limited to at our discretion no replacement or compensation product credits refund of the purchase price of or the replacement of materials that do not meet our specification By acceptance of the product Buyer indemnifies and holds MP Biomedicals harmless against and assumes all liability for the consequence of its use or misuse by the Buyer its employees or others including but not limited to the cost of handling Said refund or replacement is conditioned on Buyer notifying MP Biomedicals within thirty 30 days of receipt of product Failure of Buyer to give said notice within thirty 30 days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material s FastRNA FastDNA FastPrep and BIO 1019 Systems are registered trademarks of MP Biomedicals LLC RNApro is a trademark of MP Biomedicals LLC ys HD Application Manual Wor
23. nutes at room temperature Remove the liquid above the pellet to a new microcentrifuge tube NOTE Following centrifugation a 10 25 ul bubble may appear over a debris pellet If a bubble appears transfer only the liquid above the bubble to a new RNase free microcentrifuge tube Add 660 ul of cold 100 isopropanol to the sample invert 5 times to mix and place at 20 C for at least 30 minutes NOTE White strands may be observed in some samples The strands which include DNA and humic substances wil be removed in subsequent steps Centrifuge at gt 14 000 x g for 15 minutes at 4 and discard the supernatant NOTE The RNA pellet may appear as chocolate colored or dirty due to the presence of humic substances FastRNA Pro Soil Indirec 22 23 24 25 26 27 28 29 contamination The amount of color and contamination will vary between soil samples and will be removed in subsequent steps Carefully wash the pellet with 500 ul of cold 70 ethanol made with DEPC H O Remove the ethanol and air dry the pellet 5 minutes at room temperature NOTE DO NOT completely dry the RNA as this will increase the difficulty of resuspending the RNA in the next step Resuspend the RNA in 200 ul of DEPC H O Add 600 ul of RNAMATRIX Binding Solution and 10 ul of RNAMATRIX Slurry to the RNA Incubate at room temperature on a shaker table a rotator or with frequent inversion
24. ours at room temperature or 4 C It is best to process the soil sample through the complete protocol as soon as possible following collection 3 4 Sample Lysis with the FastPrep Instruments The fill volume in the lysing matrix tube after the addition of RNApro Solution to the sample should allow sufficient air space in the sample tube for efficient FastPrep Instrument processing Sample loss or tube failure may result from overfilling the matrix tube The matrix tube caps must be secure but not over tightened to prevent sample leakage If the sample is too large for processing in a single tube divide the sample and process using multiple tubes MP Biomedicals Lysing Matrix particles and tubes have been rigorously tested and validated in the FastPrep and FastPrep 24 Instruments The use of non MP Biomedicals products with the FastPrep Instruments is not recommended and may result in sample loss or instrument failure A single 40 second run at a speed setting of 6 0 in the FastPrep or FastPrep 24 Instrument is sufficient to lyse cells organisms and tissue present within a soil sample If the user determines that additional processing time is required the sample should be incubated on ice in the Lysing Matrix E tube for at least 2 minutes between successive FastPrep Instrument homogenizations to prevent sample over heating and possible RNA degradation 3 5 RNA Purity Humic Substance and Inhibitor Removal and Downstream
25. p 4 Add 5 ml of usersupplied Resuspension Solution either deionized H O 10 mM Tris pH 7 0 80 or 100 mM NaCl NOTE Maintain a ratio of gram soil to 5 ml Resuspension Solution Larger soil amounts may be processed in a larger tube Mix well and incubate for 10 minutes at room temperature on a rocking device or with frequent inversion to facilitate the dissociation of organic materials from soil particles Centrifuge 5 minutes at less than 500 rpm approximately 20 x g at room temperature to pellet soil and large particles NOTE Centrifugation is intended to remove the bulk of soil components from solution while permitting microorganisms to remain in solution Depending on soil type a lower speed may be used Filter the supernatant through cheesecloth VWR Grade 10 to a new centrifuge tube capable of withstanding a force of 1 800 x g FastRNA Pro Soil Indirec NOTE Cheesecloth filtration is necessary in order to remove soil particles that may have carried over from the previous centrifugation DO NOT filter through sterile gauze Kim Wipe tissue or commercially available coffee filters as SIGNIFICANT reduction in RNA yield will occur Centrifuge at 5000 rpm approximately 1 800 x g for 5 minutes at room temperature to pellet microorganisms and organic material for RNA purification Decant the supernatant and add ml of RNApro Solution to the tube Completely resuspend the pelleted materia
26. rom soil particles centrifuged and homogenized by the FastPrep or FastPrep 24 Instrument in impact resistant 2 ml tubes containing Lysing Matrix E Total RNA is released into a protective solution extracted precipitated and purified from inhibiting substances with the proprietary RNAMATRIX and optional Quick Clean Spin Filter Total RNA prepared with this kit is suitable for RT PCR and other applications 11 2 Other Related Products Description Size FastPrep 24 Instrument 00 230V FastPrep FP 00A Instrument 00V FastPrep FP 20A Instrument 120V FastPrep FP220A Instrument 220V FastRNA Pro Soil Direct Kit 50 preps FastRNA Pro Red Kit Yeast 50 preps FastRNA Pro Green Kit Plant amp Animal 50 preps FastRNA Pro Blue Kit Bacteria 50 preps FastDNA Kit 100 preps FastDNA SPIN Kit 100 preps FastDNA SPIN Kit for Soil 50 preps FastProtein Blue Matrix 50 preps FastProtein Red Matrix 50 preps RNase Erase 500 ml Catalog 6002 500 6001 100 6001 120 6001 220 6070 050 6035 050 6045 050 6025 050 6540 400 6540 600 6560 200 6550 400 6550 600 2440 204 27 28 12 Product Use Limitation amp Warranty The products presented in this instruction manual are for research or manufacturing use only They are not to be used as drugs or medical devices in order to diagnose cure mitigate treat or prevent diseases in humans or animals either as part of an accepted course of therapy or in experime
27. ss of RNA required resuspend the RNA in DEPC H O and measure the concentration by OD before proceeding NOTE RNA is generally stable for up to a year at 70 to 80 C For longer term storage RNA samples may be stored as ethanol precipitates When stored as an ethanol precipitate the RNA must be spun down washed and resuspended in aqueous solution prior to use Avoid frequent sample freeze thaw by storing isolated RNA as aliquots NOTE RNA does not evenly distribute in ethanol and can lead to inconsistent RNA amounts between samples when equal volumes are pipetted Vortex the RNA ethanol solution to disperse the RNA prior to removing the sample In situations where precise amounts of RINA are required it is best to precipitate the total amount or excess of RNA required resuspend the RNA in DEPC H O and measure the concentration by OD before proceeding 6 Optional Centrifugation Through Quick Clean Spin Filters The FastRNA Pro Soil Indirect Kit is designed to remove reverse transcription and PCR inhibitors to allow amplification of undiluted RNA for the vast majority of soil types It has been determined that in some instances RNA dilution 1 3 1 5 or 1 10 may result in greater yield of PCR product It is important to recognize that some soil samples may not RT PCR amplify due to purification of small amounts of total or target RNA that result from a low organism content or the soil sample may be exceptionally h
28. struments 9 35 RNA Purity Humic Substance and Inhibitor Removal and Downstream Applications 9 Safety Precautions 2 ea IO Basic Protocol for Al Soi EEN el Optional Centrifugation through Quick Clean Spin Dier zz Example Data Total RNA Isolation and RT PCR TPOUBIESMOOUNG iii i i iii iii 8 1 Lower Than Expected or No RNA Yield 82 Suspected RNA Degradation 8 3 Properties of the RNA Pellet 84 Genomic DNA Contamination mn 85 REPOR IMAI HO asis 8 6 Mucopolysaccharide Carbohydrate Contamination Recommended Reference Format for Publication 1 1 FastRNA Pro Sol Direct Kit Cat 6070 050 1 2 Other Related Products 12 Product Use Limitation amp Warranty Introduction to the FastRNA Pro Soil Indirect Kit and the FastPrep Instruments The FastRNA Pro Soil Indirect Kit quickly and efficiently isolates total cellular RNA from microorganisms and other specimens found in soil It is designed for use with the FastPrep and FastPrep 24 Instruments high speed benchtop devices that use a unique optimized motion to homogenize samples by multidirectional simultaneous impaction with lysing matrix particles FastPrep Instruments provide an extremely quick and highly reproducible homogenization that surpasses traditional lysis methods using enzyme dig
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