FastRNA® Pro Red Kit
Contents
1. Centrifuge the tubes at a minimum of 12 000 x g for 5 minutes at 4 C Transfer the upper phase without disturbing the interphase to a new microcentrifuge tube Add 500 ul of cold absolute ethanol invert 5X to mix and store at 20 C for at least 30 minutes Centrifuge at a minimum of 12 000 x g for 15 minutes at 4 C and remove the supernatant Wash the pellet with 500 ul of cold 75 ethanol made with DEPC H O Remove the ethanol air dry 5 minutes at room temperature DO NOT completely dry the RNA and resuspend the RNA in 100 ul of DEPC H O FastRNA Pro Red Kit 13 14 Incubate 5 minutes at room temperature Determine the RNA concentration a Dilute 5 ul of RNA into 495 ul of DEPC H O b Read the OD using as a blank c Calculate the sample ug RNA per ml using the formula 2 40 ug ml per OD 100 dilution factor ug RNA per ml Aliquot and store the RNA solution at 70 C RNA integrity can be analyzed visually using denaturing or non denaturing 2 agarose gel electrophoresis See Figure 1 6 Detailed Protocol For Yeast Cells in Culture Dilute ml of an overnight yeast culture into 14 ml of fresh media in a sterile 50 ml tube or 250 ml flask Incubate for 4 6 hours at 37 C with shaking at 150 200 rpm to reach an 09 1 0 Note The relationship between OD and cell concentration varies between species As a guideline fo2. 5 Lithium Chloride Precipitation Lithium chloride LiCl may be used to precipitate RNA while excluding carbohydrate DNA and proteins including transcription inhibitors Lithium chloride has historically been used to precipitate RNA greater than 300 nucleotides from tRNA and 55 RNA Lithium chloride precipitation may be incorporated into the FastRNA Pro Red Kit procedure Following ethanol precipitation of the RNA and resuspension in 100 ul DEPC H O add lithium chloride to a final concentration of 2 3 M e g 0 2 volumes 20 ul RNase free 8 M lithium chloride Add 2 5 volumes RNase free absolute ethanol 250 ul Mix the solution and store on ice at least 2 hours Centrifuge for 15 minutes at a minimum of 12 000 rpm at 4 C Remove the supernatant and wash the pellet with 7526 cold RNase free ethanol The ethanol wash step is critical to prevent LiCl inhibition of cell free translation and in vitro transcription Air dry and resuspend the RNA in 100 ul DEPC H O FastRNA Pro Red Kit 8 Recommended Reference Format for Publications Total RNA was isolated from cells of yeast using the FastRNA Pro Red Kit MP Biomedicals Irvine CA and the FastPrep 24 Instrument MP Biomedicals Irvine CA Samples have been homogenized for seconds at a speed setting of 9 References Molecular Cloning Sambrook and Russell Cold Spring Harbor Laboratory Press 3rd Edition 2001 2 Current Protocols in Molecular Biology John
3. O is generally stable for up to a year at 80 C For longer term storage RNA samples may be stored at 20 C as ethanol precipitates Ethanol precipitates must be pelleted and the RNA resuspended in aqueous solution prior to use NOTE RNA does not evenly distribute in ethanol and can lead to inconsistent RNA amounts between samples when equal volumes are pipetted In situations where precise amounts of RNA are required it is best to precipitate the total amount of RNA required resuspend the RNA in DEPC H O and measure the concentration by before proceeding Incubate 5 minutes at room temperature to facilitate RNA resuspension Determine the RNA concentration a Dilute 5 ul of the purified RNA into 495 ul of DEPC H O b Read the OD 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 0 2 and 0 8 If the OD reading is below the 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 Aliquot and store the RNA solution at 70 C The RNA integrity and an estimation of yield can be determined by analyzing a portion of the RNA sample using gel electrophoresis Add ug RN
4. A in 9 ul DEPC H O heat to 65 C for 5 minutes add gel loading buffer and load the sample on a 2 agarose gel containing 2 2M formaldehyde in MOPS buffer The sample is run at 80 volts for 30 minutes Ethidium bromide may be added to the denatured RNA sample at Oug 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 ribosomal 13 FastRNA Pro Red Kit RNAs as sharp distinct bands 1 8S 2 0kb and 265 3 8kb for yeast Heterogeneous sized messenger RNA may appear as a diffuse ethidium staining between and below the ribosomal bands Small RNA species such as tRNA and 5S RNA may be present in varying amounts at the dye front Figure Yeast and fungal total RNA extracted with the FastRNA Pro Red Kit Approximately 2 of the total RNA isolated from 100 mg tissue or 1010 cells was loaded onto a 1 2 denaturing agarose gel IXMOPS Lane 1 S pombe Lane 2 S cerevisiae Lane 3 pastoris Lane 4 C albicans Lane 5 common mushroom Lane 6 small common mushroom Lane 7 0 24 9 5kb RNA Ladder 7 Troubleshooting 7 1 Degraded RNA or Lower than Expected RNA Yields RNA purified using the FastRNA Pro Red Kit and analyzed by denaturing or non denaturing agarose gel electrophoresis will appear as 2 distinct ribosomal RNA rRNA bands of approximately equal fluorescent
5. G TT nonien ns 14 7 Degraded RNA or Lower than Expected Yields 14 7 2 No Pellet after Ethanol Precipitation 16 7 3 Genomic DNA Contamination E 16 7 4 Mucopolysaccharide Carbohydrate Contamination s 17 7 5 Lithium Chloride Precipitation 17 8 Recommended Reference Format for Publication 18 J E Saan pH 18 10 Related i t bett teh pes 19 Product Use Limitation amp Warranty sssssrisisisssrsrsriisisrsrsrensen 20 I Introduction to the FastRNA Pro Red Kit and the FastPrep Instruments The FastRNA Pro Red Kit is a single reagent extraction method designed to quickly and efficiently isolate total cellular RNA from yeast and fungus The RNApro Solution included in the kit is designed to efficiently inactivate cellular RNases during cell lysis to prevent RNA degradation During use the RNApro Solution is mixed with the sample in a tube containing a specifically selected lysing matrix The tube is then processed in the FastPrep or FastPrep 24 Instrument for 40 seconds to release the total cellular RNA DNA and proteins Following the FastPrep homogenization the RNA is purified and isolated by chloroform extraction and ethanol precipitation The purified RNA is ready for downstream applications including RT PCR and northern analysis The average RNA yield from 10 9 yeast cells is greater than 45 ug The FastPrep and Fast
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7. Prep 24 Instruments are 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 FastPrep Instruments in combination with FastPrep kits permit the release and purification of intact DNA RNA and proteins from virtually any source including yeast fungi bacteria spores plant seeds and leaves animal tissue organs and blood etc FastRNA Pro Red Kit 2 Kit Components and User Supplied Materials 2 1 FastRNA Pro Red Kit Components RNApro Solution x 55 milliliter bottle DEPC H O x 15 milliliter bottle Lysing Matrix C 50 x 2 milliliter tubes Short protocol each User manual each MSDS each Certificate of Analysis each 2 2 User Supplied Materials FastPrep or FastPrep 24 Instrument see section 10 Microcentrifuge Pipettmen RNase Erase Catalog H 2440 204 recommended Chloroform 100 ethanol 75 ethanol 1 5 or 2 0 ml RNase free microcentrifuge tubes Agarose Gel loading dye and RNA size marker 3 Important Considerations before Use 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 RNa
8. Wiley amp Sons Inc 2002 www currentprotocols com 10 Related Products Description Size FastPrep 24 Instrument 100 230V FastPrep FPIOOA Instrument 100V FastPrep FPI20A Instrument 120V FastPrep FP220A Instrument 220V FastRNA Pro Blue Kit 50 preps Bacteria FastRNA Pro Green Kit Plant amp Animal 50 preps FastDNA Kit O0preps 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 Lysing Matrix C 50 x 2ml tubes Lysing Matrix C 100 x 2ml tubes Lysing Matrix C 500 x 2ml tubes Catalog 6002 500 6001 100 6001 120 6001 220 6025 050 6045 050 6540 400 6540 600 6560 200 6550 400 6550 600 2440 204 6912 050 6912 100 6912 500 FastRNA Pro Red Kit 20 11 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 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 Biom
9. edicals 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 E Application Manual Worldwide Headquarters 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 M
10. ei Application Manual FastRNA Pro Red Kit Rapid Isolation of Total RNA from Yeast One Call and Fungus Using the FastPrep and FastPrep 24 Instruments Catalog 6035 050 One Source 50 Preps Storage Refrigerated or ambient temperature 4 C or 15 30 C A World of Revision 4 6035 050 06APR Biotechnol 09y DO NOT expose RNApro Solution to light for extended periods of time Reagent e n tne original in the closed kit box www mpbio com MP Biomedicals e 29525 Fountain Parkway e Solon OH 44139 e tel 1 800 854 0530 fax 1 800 334 6999 formerly e FastRNA Pro Red Kit FastRNA Pro Red Kit Rapid Isolation of Total RNA from Yeast and Fungus Using the FastPrep and FastPrep 24 Instruments Catalog 6035 050 50 Preps Storage Refrigerated or ambient temperature 4 C or 15 30 C Revision 6035 050 06APR DO NOT expose RNApro Solution to light for extended periods of time Store in the original bottle in the closed kit box FastRNA Pro Red Kit TABLE OF CONTENTS Introduction to the FastRNA Pro Red Kit and the FastPrep Instruments EE 2 Kit Components and User Supplied Materials 2 FastRNA Pro Red Kit Components sss 2 2 User SUPE Materials itat 6 3 Important Considerations before Use 6 the SATS EY PReCauBIOnS optimc Ro DR 8 5 Quick Protocol for Experienced Users e 8 6 Detailed Protocol ntes 10 T WOUBIESROOUIN
11. ent to prevent skin contact e g gloves lab coat and eye protection and prevent inhalation of reagent vapors and consumption of liquid during use Consult the enclosed Material Safety Data Sheet for additional details 5 Quick Protocol for Experienced Users For Yeast Cells in Culture Dilute ml of an overnight yeast culture into 14 ml of fresh media in a sterile 50 ml tube and incubate for 4 6 hours to reach an OD 09 1 0 Remove 0 ml of the culture to a 15 ml conical tube and pellet the cells by centrifugation Decant the supernatant and add ml of RNApro solution to the tube and resuspend the cells by pipetting or vortexing Transfer ml of the resuspended mixture to a red cap tube containing Lysing Matrix C provided in the kit For Cell Pellets or Fungal Tissue Add ml of RNApro Solution to a red cap tube containing Lysing Matrix C provided in the kit Add 100 mg fungal tissue or pelleted cells to the sample tube Process the tube in the FastPrep or FastPrep 24 Instrument for 40 seconds at a setting of 6 0 Remove and centrifuge the tube at a minimum of 2 000 x g for 5 minutes at 4 C Transfer the upper phase 750 ul to anew microcentrifuge tube Avoid transferring the debris pellet and lysing matrix Incubate the transferred sample 5 minutes at room temperature Add 300 ul of chloroform NO isoamyl alcohol Vortex 10 seconds and then incubate 5 minutes at room temperature
12. he solution surface Recentrifuge the sample in the same tube and exercise caution to not lose the pellet when removing the supernatant Confirm enough sample was used to isolate RNA Since many differences exist between yeast strains and fungal species it may be necessary to increase the amount of starting material in order to recover the desired amount of RNA The relationship between cells per milliliter and OD igo reading is not exact but OD ooo is generally between 10 and 10 cells per milliliter 7 3 Genomic DNA Contamination Genomic DNA contamination will appear as a high molecular weight smear on a denaturing gel or as ethidium bromide stained material in the gel loading well In the event genomic DNA contamination occurs re extract the RNA sample with chloroform or chloroform isoamyl alcohol 24 1 vv The lower phase of the chloroform extraction contains genomic DNA and should be carefully avoided when removing the top RNA containing phase Leaving a small volume of the top phase in the tube will prevent accidental DNA contamination 7 4 Mucopolysaccharide Carbohydrate Contamination Samples containing large amounts of cellular mucopolysaccharides can be re extracted after the initial chloroform extraction with a second chloroform extraction Isoamyl alcohol may be included with the chloroform CHCI3 IAA 24 1 v v to increase RNA purity Refer also to Lithium Chloride Precipitation in the Troubleshooting section 7
13. intensity using ethidium bromide staining Messenger RNA mRNA which typically represents approximately less than 1 of the total cellular RNA and is heterogeneous length will not be visible as distinct bands rRNA is used as a marker to assess sample RNA degradation Degraded RNA may appear as unequal fluorescent intensity between bands a single band may be completely lacking or a heterogeneous fluorescent smear may appear below the rRNA bands or throughout the gel lane Recommended precautions include cleaning all instruments and work area with RNase Erase Catalog 2440 204 prior to use Use disposable sterile plastic containers when possible Glassware should be thoroughly cleaned rinsed with DEPC H O and baked at 250 C for 4 hours to remove RNase Sterile plugged micropipettes are recommended see 2 for additional suggestions Certain samples may contain elevated RNase levels Reduce the exposure time to RNase by adding RNApro Solution to each sample as soon as possible following sample harvest Process fewer samples to shorten the time before complete cellular lysis and exposure to the RNase inactivating activity of RNApro Solution Yeast cells in log phase growth with maximal aeration and nutrients provide the highest yield and integrity RINA Yeast cells in stationary phase growing in oxygen or nutrient limiting conditions stored for extended duration at room temperature or refrigerated for extended periods wil
14. l contribute to reduced RNA yield and integrity RNApro Solution can permeate samples and will protect RNA from degradation for at least 24 hours before it is processed in the FastPrep Instrument However higher yields of RNA will always result when samples are homogenized immediately after the addition of RNApro Solution 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 reagents FastRNA Pro Red Kit RNA degradation may occur due to RNase contamination introduced into the DEPC H O following use If contamination is suspected prepare fresh DEPC H O in an RNase free container 1 2 RNApro Solution contains RNase inactivating components and will not support active RNase contamination 7 2 No Pellet after Ethanol Precipitation The purified RNA may not appear as a pellet but may instead adhere to the side of the tube The RNA may not be visible and it may appear that RNA has not been purified Complete the RNA purification 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 The RNA pellet may not be firmly attached to the side of the tube and may be observed floating in the solution or at t
15. ld Add 300 ul of chloroform NO isoamyl alcohol Vortex 10 seconds Incubate 5 minutes at room temperature to permit nucleoprotein dissociation and increase RNA purity Centrifuge the tubes at a minimum of 12 000 x g for 5 minutes at 4 C FastRNA Pro Red Kit 10 Transfer the upper phase to a new microcentrifuge tube without disturbing the interphase If a portion of the interphase is transferred repeat the centrifugation with the upper phase and transfer the new upper phase to a clean microcentrifuge tube NOTE Samples containing large amounts of cellular mucopolysaccharides can be re extracted with chloroform isoamyl alcohol may be included with the chloroform CHCI3 IAA 24 1 v v to increase RNA purity Alternatively a lithium chloride precipitation may be used see the Troubleshooting section Add 500 ul of cold absolute ethanol to the sample invert 5X to mix and store at 20 C for at least 30 minutes Centrifuge at a minimum of 12 000 x g for 15 minutes at 49C and remove the supernatant The RNA will appear as a white pellet in the tube If the pellet is floating the sample may be recentrifuged to place the pellet at the tube bottom Wash the pellet with 500 ul of cold 75 ethanol made with DEPC H O Remove the ethanol air dry 5 minutes at room temperature DO NOT completely dry the RNA and resuspend the RNA in 100 ul of DEPC H O for short term storage RNA resuspended in DEPC H
16. ol may be included with the chloroform CHCI3 IAA 24 1 v v extraction after Step 8 Quick Protocol for Experienced Users or in step 10 Detailed Procedure to reduce the potential carryover A single 40 second run at a speed setting of 6 in the FastPrep or FastPrep 24 Instrument is sufficient to lyse most yeast or fungal samples If the user experimentally determines that additional processing time is required the sample should be incubated on ice in the Lysing Matrix tube for at least 2 minutes between successive FastPrep Instrument homogenizations to prevent sample heating and possible RNA degradation The FastRNA Pro Red Kit is designed to selectively purify total cellular RNA from DNA and protein Experiments have indicated FastRNA Pro Red Kit the RNA is sufficiently pure for use in RT PCR and Northern analysis however it is recommended the user incorporate DNase treatment of the RNA prior to use in applications where absolute control of DNA contamination is essential Use DNase at the concentration recommended by the manufacturer and incubate at 37 C for 30 minutes The 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 4 Safety Precautions The RNApro Solution contains components that when in contact with human tissue or during inhalation may cause irritation or burning Wear personal protective equipm
17. r yeast use 1 0 OD is X 10 cells per milliliter Remove 10 ml of the culture to a 15 ml conical tube and pellet the cells by centrifugation at 2 800 rpm x 1 500 g for 5 minutes at 4 C e g Beckman ModelT 6 Centrifuge l 92 Swinging Bucket Rotor for 10 minutes Decant the supernatant and add ml of RNApro Solution to the tube Completely resuspend the cells by pipetting or vortexing Transfer ml of the resuspended mixture to a red cap tube containing Lysing Matrix C provided in the kit For Cell Pellets or Fungal Tissue Add ml of RNApro Solution to a red cap tube containing Lysing Matrix C provided in the kit Add 100 mg fungal tissue or pelleted cells to the sample tube Securely close the cap ofthe Lysing Matrix C tube to prevent leakage during homogenization NOTE The calculated volumes will provide adequate airspace in the matrix tube to prevent sample leakage and or tube failure DO NOT overfill the matrix tube To process a greater number of cells or larger sample use a second matrix tube Process the sample tube in the FastPrep or FastPrep 24 Instrument for 40 seconds at a setting of 6 0 Remove the sample tube and centrifuge at a minimum of 12 000 x g for 5 minutes at 4 C or room temperature Transfer the upper phase to a new microcentrifuge tube Avoid transferring the debris pellet and lysing matrix Incubate the transferred sample 5 minutes at room temperature to increase RNA yie
18. se contamination by using gloves throughout the procedure using DEPC H O and by treating pipettmen work area gel box and gel comb with RNase Erase Additional RNA handling methods and precautions may be found in references and 2 The volume after the addition of RNApro Solution to the sample has been calculated to maintain a sufficient air space in the sample tube during FastPrep Instrument processing Sample loss or tube failure may result from overfilling the matrix tube The matrix tube caps must be secure but not overtightened to prevent sample leakage If the sample is too large for processing in a single tube divide the sample and process using multiple tubes Confirm the sample tubes spin freely and will not scrape the microcentrifuge wall during centrifugation The use of other manufactured tubes in the FastPrep Instruments is not recommended and may result in sample loss or instrument failure Add the RNApro Solution to the sample as soon as possible to initiate RNase inhibition Samples both FastPrep Instrument homogenized and non homogenized are stable in RNApro Solution overnight at room temperature or 4 C Yeast and fungus strain variability may result in unwanted protein and mucopolysaccharide carryover into the aqueous solution following chloroform extraction While this may not compromise downstream applications the user may adapt the protocol to include an additional chloroform isoamyl alcoh
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