FastRNA® Pro Soil
Contents
1. 29 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 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 Direct 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 15 FastRNA Pro Soil Direct of small amounts of total or target RINA that result from a low organism content or the soil sample may be exceptionally high in inhibiting substances incl2. Mucopolysaccharide Carbohydrate Contamination Cellular mucopolysaccharides will not co purify with RNA using the protocol and reagents in the FastRNA Pro Soil Direct Kit 9 Recommended Reference Format for Publication Total RNA was isolated from g of type soil using the FastRNA Pro Soil Direct 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 199 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
3. 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 RNA Finger tap the tube bottom frequently to provide gentle mixing Microcentrifuge pulse spin approximately 0 seconds and transfer the supernatant containing eluted RNA to a new tube NOTE Do not discard the RNAMATRIX pellet Repeat step 24 and 25 to provide a final RNA volume of 100 ul NOTE If matrix carryover occurs remove the matrix by pulse spinning the microcentrifuge tube for approximately 10 seconds to pellet the matrix Carefully transfer only the supernatant to a new RNase free microcentrifuge tube FastRNA Pro Soil Dir 27 Determine the RNA concentration and integrity a Dilute 5 ul of the purified RNA into 495 ul of DEPC H O b Read the OD260 using DEPC H O 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 02 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 electr
4. from 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 100 230V FastPrep FP 100A Instrument 00V FastPrep FP120A Instrument 120V FastPrep FP220A Instrument 220V FastRNA Pro Soil Indirect 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 00 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 6075 050 6035 050 6045 050 6025 050 6540 400 6540 600 6560 200 6550 400 6550 600 2440 204 25 FastRNA Pro Soil Dire 26 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 o
5. of RNA and DNA from soils and sediments Appl Environ Microbiol 67 4495 503 23 24 Ogram A G S 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 71 Humus Chemistry Stevenson F J 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 l I Related Products 11 1 FastRNA Pro Soil Indirect Kit Cat 6075 050 The FastRNA Pro Soil Indirect Kit is designed to efficiently isolate total RNA from organic material found in soil samples Cellular material is washed away
6. C ys HD Application Manual Worldwide Headguarters MP Biomedicals Gmbh MP Global d o o Tel 1 440 337 1200 Phone 0800 426 67337 Tel 381 11 2622 945 Toll Free Tel 800 854 0530 Fax 0800 629 67337 Fax 381 11 2623 373 Fax 1 440 337 1180 Toll Free Fax 800 334 6999 MP Biomedicals Italy MP Biomedicals Asia Pacific Pte Ltd Tel 0800 011 643 Tel 65 6775 0008 Toll Free Phone 00800 7777 9999 Fax 0800 255 220 Fax 65 6775 4536 Toll Free Fax 00800 6666 8888 MP Bio Japan K K MP Biomedicals Switzerland MP Biomedicals Australasia Pty Ltd Tel 03 3808 2102 Tel 061 271 0007 Tel 61 2 9838 7422 Toll Free Tel 0120 788 020 Fax 061 271 0084 Fax 61 2 9838 7390 Fax 03 3808 2401 MP Biomedicals FZ LLC MP Biomedicals Europe n v s a MP Biomedicals Netherlands Tel 971 4 367 6544 Tel 02 466 00 00 Tel 0800 0227416 Fax 971 4 368 8031 Fax 02 466 26 42 Fax 0800 0227489 MP Biomedicals UK MP Biomedicals Canada MP Biomedicals Poland Tel 0800 282 474 Tel 888 362 5487 Tel 48 22 659 58 95 Fax 0800 614 735 Fax 514 935 7541 Fax 48 22 658 45 05 MP Biomedicals France MP Biomedicals Russia Tel 03 88 67 54 25 Tel 7 095 995 2844 Fax 03 88 67 19 45 Fax 7 095 995 2846 MP Biomedicals e 29525 Fountain Parkway e Solon OH 44139 e tel 1 800 854 0530 fax 1 800 334 6999 formerly 4 Q BlOgene e
7. TRIX Binding Solution 35m RNAMATRIX Wash Solution Concentrate I5 m DEPC Treated H O 30 m Lysing Matrix E 50 tubes Quick Clean Spin Filters 50 filters Catch Tubes 50 tubes User Manual each MSDS each Certificate of Analysis each 2 2 User Supplied Materials FastPrep or FastPrep 24 Instrument See Section Related Products RNase Erase See Section Related Products 100 Ethanol Microcentrifuge Chilled 7076 Ethanol prepared with DEPC treated H O Chilled Isopropanol 1 5 or 2 0 ml RNase free Microcentrifuge Tubes Agarose Gel Loading Dye Electrophoresis Size Marker 3 Important Considerations before Use 3 1 Preparation of RNAMATRIX Wash Solution The FastRNA Pro Soil Direct 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 3 2 Preparing to Isolate Total RNA The presence or introduction of RNase during the procedure may result in sample degradation It is strongly recommended FastRNA Pro Soil Dire that the user minimize the potential for RNase contamination by wearing gloves throughout the procedure using DEPC H O and by treating pipettors 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 14 Confirm the Lysi
8. and Downstream Applications Safety Precautions r Basic Protocol forAl Soil ae saat zd Optional Centrifugation through Quid Clean Spin DS wc Example Data Total RNA Isolation and RT PCR 7 page 18 8 1 Lower Than Expected or No RNA Yield 18 8 2 Suspected RNA Degradation 19 8 3 Properties of the RNA Pellet m 20 84 Genomic DNA Contamination sdl 8 5 RT PCR Inhibition 8 6 Mucopolysaccharide Carbohydrate ContaminatioN 22 Recommended Reference Format for Publication Related Bebe r pe 1 1 FastRNA Pro Sol SES Cati 6075 050 win D 1 2 Other Related Products Product Use Limitation amp Warranty I Introduction to the FastRNA Pro Soil Direct Kit and the FastPrep Instruments The FastRNA Pro Soil Direct 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 digestion sonication blending douncing and vortexing Soil biodiversity is directly affected by its physical and chemical c
9. e Application Manual Rapid Isolation of Total RNA from Soil Using the FastPrep and FastPrep 24 Instruments Catalog 6070 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 6070 050 06APR MP Biomedicals e 29525 Fountain Parkway e Solon OH 44139 e tel 1 800 854 0530 e fax 1 800 334 6999 formerly e FastRNA Pro Soil Direct Kit FastRNA Pro Soil Direct Kit Rapid Isolation of Total RNA from Soil Using the FastPrep and FastPrep 24 Instruments Catalog 6070 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 6070 050 06APR FastRNA Pro Soil Direc TABLE OF CONTENTS D o Ud 12 References LE Introduction to the FastRNA Pro Soil Direct Kit and the FastPrep Instruments w n GE Kit Components and Oe Supplied Materia paanan Ud 2 FastRNA Pro Soil Direct Kit Components d 22 User Supplied Materials senn Important Considerations before Use L 3 1 Preparation of RNAMATRIX9 Wash Schon 32 Preparing to Isolate Total RINA 3 3 Sample Lysis with the FastPrep betr ment 8 34 RNA Purity Humic Substance and Inhibitor Removal
10. f an accepted course of therapy or in experimental 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 LL
11. gned to remove genomic 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 v v 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 Direct 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 Section 5 provides the first level of purification using proprietary RNAMATRIX Section 6 provides second level purification through Quick 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 purification It has been demons
12. is used as a marker to assess sample degradation Degraded RNA or mRNA may appear as 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 FastRNA Pro Soil Dir 20 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 Nase may have been introduced during isolation To prevent NAse contamination the use of gloves RNase free plugged Dette tips and RNase free tubes is strongly recommended Clean petors and work area with RNase Erase Catalog 2440 204 rior to beginning RNA isolation Use DEPC treated reagents NApro Soil Lysis Solution already contains RNase inactivating omponents and will not support RNase contamination nsure that DEPC treated H O was used to make the 70 thanol Artifactual RNA degradation may occasionally occur during gel electrophoresis from a gel that was not RNase free running the gel at too high voltage or using depleted running buffer Rerun the samples with a known intact RNA sample using freshly prepared RNase free electrophoresis reagents 2Y V V D P o mo 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 no
13. mple tube containing Lysing Matrix E NOTE The number of organisms and the amount of RNA available for isolation will vary among soil samples and is related to the amount of moisture chemical and physical composition endogenous organism population and environmental conditions at the time of collection The greatest RNA yield is obtained in freshly collected soil that is not stored for extended period of time If the soil is nown to contain few microorganisms or if the RNA yield is expected to be low the amount of soil processed may be increased using additional lysing matrix tubes and by pooling the purified RINA Be sure to leave adequate airspace 250 500 yl in the matrix tube to prevent sample leakage and or tube failure DO NOT overfill the matrix tube To process a greater amount of soil at one time the FastRNA Pro Soil Indirect Kit may be used See Section Related Products Add ml of RNApro Soil Lysis Solution to the tube Invert several times to resuspend the soil and lysing matrix in the solution Ensure the cap is securely closed to prevent leakage in the next step Process the tube in the FastPrep or FastPrep 24 Instrument for 40 seconds at a setting of 6 0 Remove the tube and centrifuge at 214 000 x g for 5 minutes at room temperature Transfer the liquid to a new microcentrifuge tube NOTE Some debris carryover will not affect subsequent steps Add 750 ul of Phenol Chloroform 1 1 and vo
14. ng Matrix E tubes spin freely and will not scrape the microcentrifuge wall during centrifugation Add RNApro Soil Lysis Solution to the sample as soon as possible after sample collection to initiate RNase inhibition FastPrep Instrument homogenized and non homogenized samples are stable in RNApro Soil Lysis Solution for up to 24 hours at room temperature or 4 C lt is best to process the soil sample through the complete protocol as soon as possible following collection 3 3 Sample Lysis with the FastPrep Instruments The fill volume in the lysing matrix tube after the addition of RNApro Soil Lysis Solution to the sample should allow sufficient air space in the sample tube for efficient FastPrep Instrument processing Add as much soil as possible to the RNApro Soil Lysis Solution and Lysing Matrix E while allowing between 250 500 ul of empty space in the tube Sample loss or tube failure may result from overfiling 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 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
15. omposition 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 I 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 direct method consists of extracting nucleic acid from microorganisms and other biological specimens directly from soil 7 The indirect method utilizes an initial buffer based separation of microorganisms and other biological specimens from the soil followed by lysis of the organisms and nucleic acid purification 8 9 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 FastRNA Pro Soil Kits are available from MP Biomedicals for both direct 6070 050 and indirect 6075 050 RNA isolation methods FastRNA Pro Soil Dire Soil types differ in the type and amount of organic materials The largest and most chemically significant fraction of natural organic matter is the humic subs
16. ophoresis to approximate the concentration 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 Run 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 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 28 Evaluate the purified RNA for use in RI 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
17. resence of humic substances contamination The amount of color 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 NOTE The RNA may be pipetted to enhance resuspension 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 for 5 minutes 20 21 22 23 24 25 26 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 10 seconds and discard the supernatant Use caution not to remove the RNAMATRIX Microcentrifuge pulse spin a second time for approximately 10 seconds and carefully remove any residual wash solution with a pipet Use caution not to remove the RNAMATRIX Air dry 5 minutes
18. rom 0 5 g of four different soil samples was loaded on to a 0 8 agarose gel Lane kb ladder Lane 2 Soil 1 Lane 3 Soil 5 Lane 4 Soil 10 Lane 5 Soil 11 FastRNA Pro So PICTURE 2 FastRNA Pro Soil Direct Kit RFPCR of Fungal Gene from Total RNA Isolated from Soil Samples with the FastRNA Pro Soil Direct Kit Approximately 40 of the RT PCR reaction was loaded on to a 0 8 agarose gel Lane 50bp 2kb marker Lane 2 Soil 1 Lane 3 Soil 5 Lane 4 Soil 10 Lane 5 Soil 11 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 To process a greater amount of material at one time the FastRNA Pro Soil Indirect Kit may be used See Section Related Products 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 the stability sample
19. rtex 10 seconds NOTE Do not use Phenol Chloroform other than that supplied with the kit Inconsistent results and poor RNA yield may occur using non kit reagents 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 upper aqueous phase to a new centrifuge tube without disturbing the interphase NOTE Ifa portion ofthe interphase is accidentally transferred repeat the centrifugation in step 8 with the contaminated upper phase and transfer the new upper phase to a clean microcentrifuge tube Add 200 ul of Inhibitor Removal Solution Invert 5 times to completely mix Centrifuge at 2 14 000 x g for 5 minutes at room temperature Remove the liquid above the pellet to a new microcentrifuge tube Centrifuge at 14 000 x g for 15 minutes at 4 C and discard 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 10026 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 the supernatant NOTE The RNA pellet may appear as chocolate colored or dirty due to the p
20. s over extended time periods e g 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 control tube indicates the soil stability sample RNA was likely degraded during soil storage prior to the addition of RNApro Soil Lysis Solution Certain bacterial strains may contain elevated RNase levels Reduce the exposure time to RNase by adding the RNApro Soil Lysis Solution to each sample as soon as possible following sample harvest RNApro Soil Lysis Solution will protect RNA in soil samples from degradation for at least 24 hours at room temperature or 49C 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 126 of the total cellular RNA and is heterogeneous length may not be highly visible Ribosomal RNA
21. setting of 6 0 in the FastPrep or FastPrep 24 Instrument is sufficient to lyse cells organisms and tissues 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 4 RNA Purity Humic Substance and Inhibitor Removal and Downstream Applications The FastRNA Pro Soil Direct 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 The FastRNA Pro Soil Direct 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 and to allow amplification of undiluted RINA for the vast majority of
22. 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 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 Clean Spin Filters provided in the kit Section 6 Centrifugation of purified RNA through the Quick Clean Spin Filter as directed will remove residual inhibitors with no significant loss of RNA quantity FastRNA Pro Soil Dire 4 Safety Precautions The RNApro Soil Lysis Solution and Phenol Chloroform Solution contain 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 Weigh 0 5 g tol O g of soil and transfer to a purple cap sa
23. t be visible in the pellet or on the tube side and it may appear that RNA has not been purified Complete the protocol and confirm the RNA concentration by OD 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 not to lose the pellet when removing the supernatant A brown color present in the RNA pellet after Step 4 is most likely due to co purification of humic substances which will be removed by the RNAMATRIX in steps 18 26 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 RI 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 RT PCR it is recommended the samples be additionally purified using the kit provided Quick Clean Spin Filters 8 4 Genomic DNA Contamination The FastRNA Pro Soil Direct Kit is desi
24. tances 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 Direct 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 RT PCR inhibitors for use in downstream applications In the event further processing is required a second level of purification through Quick Clean Spin Columns will provide additional purification of colorless and contaminant free RNA 2 Kit Components and User Supplied Materials 2 1 FastRNA Pro Soil Direct Kit Components RNApro Soil Lysis Solution 55m Phenol Chloroform 1 1 50m Inhibitor Removal Solution 12 m RNAMATRIX Slurry 0 6 ml RNAMA
25. trated that in some instances 21 FastRNA Pro Soil Dir 22 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 Direct Kit as an optional purification step Centrifugation of purified RNA through the Quick Clean Spin Filter as directed will remove residual inhibitors with no significant loss of RNA quantity See Section 6 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 also 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
26. uding 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 Clean Spin Filters provided in the kit Section 6 Centrifugation of purified RNA through the Quick Clean Spin Filter as directed will remove residual inhibitors with no significant loss of RNA quantity Ifthe RNA sample is frozen thaw completely and centrifuge briefly to collect the liquid at the bottom of the tube before proceeding 2 Apply 50 ul DEPC H O to the Quick Clean Spin Filter insert into a user supplied RNase free microcentrifuge tube and pulse microcentrifuge for 0 seconds 3 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 4 Pulse spin the Quick Clean Spin Filter and Catch Tube for 10 seconds to collect purified RNA 5 Quantify the RNA per Step 27 in Section 5 7 Example Data Total RNA Isolation and RT PCR PICTURE FastRNA Pro Soil Direct Kit Total RNA extracted from Soil Samples with the FastRNA Pro Soil Direct Kit Approximately 5 of the total RNA isolated f
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