PowerMicrobiome RNA Isolation Kit Sample (Catalog No S)

Transcription

1 PowerMicrobiome RNA Isolation Kit Sample (Catalog No S) Information for Ordering Product Catalog No. Quantity Preps Instruction Manual Inhibitor Removal Technology (IRT) is a registered trademark of MO BIO Laboratories, Inc. and is covered by the following patents USA US 7,459,548 B2, Australia , Japan and India Please recycle Version:

2 Table of Contents Introduction... 3 Protocol Overview... 3 Flow Chart... 4 Equipment Required... 5 Kit Contents & Storage... 5 Precautions & Warnings... 5 Important Notes Before Starting... 6 Protocols: Experienced User Protocol... 7 Detailed Protocol (Describes what is happening at each step)... 9 Hints & Troubleshooting Guide Contact Information Products recommended for you

3 Introduction The PowerMicrobiome RNA Isolation Kit is designed for fast and easy purification of total RNA from samples high in PCR inhibitors; including stool, gut material, dried feces, contaminated buccal swabs, and secretions. Patented Inhibitor Removal Technology (IRT) ensures complete removal of the inhibitory substances often contained in these materials. For example, undigested plant material in the gut or heme compounds from lysed red blood cells, abundant in stool. The result is RNA that is ready to use in the most demanding downstream applications. Genomic DNA is removed on column using the provided DNase I and reaction buffer. RNA is eluted in RNase-Free Water and ready for cdna synthesis and RT-qPCR. Protocol Overview The recommended starting amount is 0.25 grams of stool or biosolid. Stool samples must be stored at -80 o C as soon as possible after collection to protect the integrity of the RNA. Lysis is achieved using Glass Bead Tubes, 0.1 mm in combination with a strong chemical lysis buffer that ensures efficient extraction of tough microorganisms in the bead beating step. The binding solution (PM3) captures the total nucleic acid content in the lysate and DNA is removed by the use of on-column DNase followed by a wash solution to remove the enzyme and digested nucleic acids. The RNA is eluted in RNase-Free Water and is ready to use in RT-PCR. If additional DNase treatment is required, we recommend the RTS DNase Kit (cat# ), a novel method for removing genomic DNA using a room temperature stable DNase enzyme. With the RTS DNase, up to 30 ug of DNA is removed in 20 minutes and the DNase enzyme is easily removed using a proprietary resin. No heat or EDTA is required to inactivate the DNase. RNA retains its original quality and stability when treated with the RTS DNase Kit. This kit is for research purposes only. Not for diagnostic use. Other Related Products Catalog No. Quantity DNase Max Kit preps Vortex Adapter for 2 ml tubes V unit Sample Storage and Preservation The yield and integrity of the RNA isolated from microbes in stool is greatly influenced by the state of the digestive system, diet of the individual, and the length of time between collection of the sample and preservation at -80 o C. The main components of stool are water (between 65%-85%), undigested food, bile, bilirubin, which is derived from dead red blood cells, and dead bacterial cells (up to 50%). Because of the high content of dead and decaying bacterial and human cells, RNA isolated from stool will typically appear to have some level of degradation using standard analysis methods. Quantitative PCR assays developed for RNA species from stool or gut should be designed to detect small fragments, to increase detection sensitivity. To optimize the quality of RNA from stool, process the sample as quickly as possible after collection. It may be preferable to freeze the samples at -80 o C in small aliquots to avoid freeze/thaw cycles of the bulk sample, which can increase the lysis of cells and degradation of RNA. Samples should be processed rapidly by adding the βme containing PM1 lysis buffer to the bead tube before the sample has fully thawed. Homogenize immediately to saturate the cellular RNA in the protective lysis buffer. For fresh (non-frozen) samples, rapid homogenization in lysis buffer is especially critical in order to isolate the highest quality RNA. 3

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5 Equipment Required Microcentrifuge (13,000 x g) Pipettors Vortex-Genie 2 Vortex (MO BIO Catalog# V or V-220) Vortex Adapter (MO BIO Catalog #13000-V1-24) Reagents Required but not Included β-mercaptoethanol Optional Reagents Phenol:Chloroform:Isoamyl Alcohol (25:24:1) ph (Amresco Catalog#: , ) or (US) ) Kit Contents Components Amount Glass Bead Tubes, 0.1 mm 2 Solution PM1 1.5 ml Solution PM2 0.5 ml Solution PM3 1.5 ml Solution PM4 3 x 1 ml Solution PM5 3 x 1 ml Solution PM6 0.1 ml Solution PM7 1 ml Solution PM ml RTS DNase 15 µl Spin Filters 2 2 ml Collection Tubes 8 Kit Storage Remove the tube of RTS DNase and store at 4 C. Store all other reagents and kit components at room temperature (15-30 C). The RTS DNase may be stored at 4 C for up to two years or at room temperature for up to 6 months without loss of activity. DNase is sensitive to physical denaturation. Do not vortex the DNase. Precautions Please wear gloves when using this product. Avoid all skin contact with kit reagents. In case of contact, wash thoroughly with water. Do not ingest. See Material Safety Data Sheets for emergency procedures in case of accidental ingestion or contact. All MSDS information is available upon request ( ) or at Reagents labeled flammable should be kept away from open flames and sparks. WARNING: Solutions PM4 and PM5 contain alcohol. They are flammable. 5

6 Important Notes Before Starting Solution PM1 must be warmed at C for 10 minutes to dissolve precipitates prior to use. Solution PM1 should be used while still warm. Shake to mix Solution PM5 before use. Prepare Solution PM1/βME by adding β- mercaptoethanol (βme) to Solution PM1 Add enough β- mercaptoethanol (βme) to Solution PM1 to produce a final concentration of 10 µl/ml. For best results do not use the PM1/βME mixture if greater than a month old. Since the PM1/βME mixture loses its effectiveness over time, it s best to make it fresh each time. For each prep, you will need 650 µl of PM1/βME solution. Alternatively, you may add 650 µl of solution PM1 and 6.5 µl βme to each Glass Bead Tube, 0.1 mm. Note: Use a fume hood when opening βme to avoid exposure to the chemical. DNase I Stock Enzyme and DNase I Solution Preparation and Storage The DNase provided in this kit is the RTS DNase, a room temperature stable DNase stored as a liquid. The concentration of the RTS DNase is 10 units/µl. Before using, centrifuge the tube briefly to bring all of the DNase into the bottom of the tube. This enzyme should be stored at 4 o C for up to two years and can be kept at room temperature for up to 6 months without loss of activity. Additional RTS DNase may be purchased using catalog number To prepare the DNase I Solution, determine the volume of enzyme needed according to the number of samples. Per prep, combine 4 µl of RTS DNase I enzyme with 46 µl of Solution PM6. 6

7 Experienced User Protocol Please wear gloves at all times Warm Solution PM1 prior to use at 55 C for 10 minutes. Use Solution PM1 while still warm. 1. Place 0.25 grams of stool or biosolid sample into the Glass Bead Tube, 0.1 mm provided. Note: If a phenol-based lysis is desired, add 100 µl of phenol:chloroform:isoamy alcohol ph to the Bead Tube before adding the sample. 2. Add 650 µl of Solution PM1/βME (see Important Notes before Starting section) to the Glass Bead Tube, 0.1 mm. Alternatively, you may add separately 650 µl PM1 and 6.5 µl of βme to the Glass Bead Tube. 3. Secure the Glass Bead Tube, 0.1mm horizontally to a MO BIO Vortex Adapter, catalog number V1 or V1-24. The tube caps should be oriented pointing toward the center of the Vortex Adapter. 4. Vortex at maximum speed for 10 minutes. 5. Centrifuge at 13,000 x g for 1 minute at room temperature. Transfer the supernatant to a clean 2 ml Collection Tube (provided). The expected volume is ~ µl. If you added phenol:chloroform: isoamyl alcohol, remove the upper aqueous layer and transfer to a clean 2 ml Collection Tube. 6. Add 150 µl of Solution PM2 and vortex briefly to mix. Incubate at 4 C for 5 minutes. 7. Centrifuge the tubes at 13,000 x g for 1 minute. 8. Avoiding the pellet, transfer the supernatant to a clean 2 ml Collection Tube (provided). Do not transfer more than 650 µl at this step. 9. Add 650 µl of Solution PM3 and 650 µl of Solution PM4. Vortex briefly to mix. Note: To prevent small RNA species (5S, trna and degraded RNA) from co-purifying with the mrna and rrna, use 650 µl of 70% ethanol instead of Solution PM4. For the purification of small RNAs, such as microrna and sirna, transfer the lysate to a larger tube to accommodate a higher volume (2.6 ml) and add an additional 650 µl of 100% ethanol to the lysate. You will need to supply 100% ethanol for this step. 10. Load 650 µl of supernatant onto a Spin Filter and centrifuge at 13,000 x g for 1 minute. Discard the flow through and repeat until all the supernatant has been loaded onto the Spin Filter. Note: A total of three loads for each sample processed is required and four loads if an additional volume of 100% ethanol is added for the microrna and sirna protocol. 11. Shake to mix Solution PM5. Add 650 µl of Solution PM5 to the Spin Filter and centrifuge at 13,000 x g for 1 minute. Note: Skip steps if you want to isolate both RNA and DNA. 12. Discard the flow through and centrifuge again at 13,000 x g for 1 minute to remove residual wash. 7

8 13. Place the Spin Filter basket into a clean 2 ml Collection Tube (provided). 14. To the center of the Spin Filter, add 50 µl of DNase I Solution (prepared by mixing 46 µl of Solution PM6 and 4 µl of RTS DNase enzyme. See Important Notes Before Starting section). 15. Incubate at room temperature for 15 minutes. 16. Add 400 µl Solution PM7 and centrifuge the column at 13,000 x g for 1 minute. 17. Discard the flow through and add 650 µl of Solution PM5 and centrifuge at 13,000 x g for 1 minute. 18. Discard the flow through and add 650 µl of Solution PM4 and centrifuge at 13,000 x g for 1 minute. 19. Discard the flow through and centrifuge again at 13,000 x g for 2 minutes to remove any residual wash solution. 20. Place the Spin Filter basket into a clean 2 ml Collection Tube (provided). 21. Add 100 µl of Solution PM8 (RNase-Free Water) to the center of the white filter membrane. Allow the water to sit on the membrane for at least 1 minute. Note: Eluting with 100 µl of Solution PM8 will maximize RNA yield. For more concentrated RNA, a minimum of 50 µl of Solution PM8 can be used. Do not use less than 50 µl of Solution PM Centrifuge at 13,000 x g for 1 minute. 23. Discard the Spin Filter basket. The RNA is now ready for any downstream applications. The RNA in the tube can be stored at -80 C until use. Thank you for choosing the PowerMicrobiome RNA Isolation Kit! For further removal of genomic DNA post-rna extraction, we recommend the RTS DNase Kit, catalog # , which contains a DNase removal resin to generate DNAfree RNA without the use of heat or EDTA to inactivate enzymes. 8

9 Detailed Protocol (Describes what is happening at each step) Please wear gloves at all times Warm Solution PM1 prior to use at 55 C for 10 minutes. Use Solution PM1 while still warm. 1. Place 0.25 grams of stool or biosolid sample into the Glass Bead Tube, 0.1 mm provided. Note: If a phenol-based lysis is desired, add 100 µl of phenol:chloroform:isoamy alcohol ph to the Bead Tube before adding the sample. 2. Add 650 µl of Solution PM1/βME (see Important Notes before Starting section) to the Glass Bead Tube, 0.1 mm. Alternatively, you may add separately 650 µl PM1 and 6.5 µl of βme to the Glass Bead Tube. 3. Secure the Glass Bead Tube, 0.1 mm horizontally to a MO BIO Vortex Adapter, catalog number V1 or V1-24. The tube caps should be oriented pointing toward the center of the Vortex Adapter. 4. Vortex at maximum speed for 10 minutes. What s happening: The sample is homogenized using mechanical bead beating and a lysis buffer that protects the RNA released into the supernatant. 5. Centrifuge at 13,000 x g for 1 minute at room temperature. Transfer the supernatant to a clean 2 ml Collection Tube (provided). The expected volume is ~ µl. If you added phenol:chloroform: isoamyl alcohol, remove the upper aqueous layer and transfer to a clean 2 ml Collection Tube. What s happening: Proteins and cellular debris are pelleted with the beads and the supernatant contains RNA and DNA from both human and bacterial cells. 6. Add 150 µl of Solution PM2 and vortex briefly to mix. Incubate at 4 C for 5 minutes. What s happening: Solution PM2 is Inhibitor Removal Solution which completes the IRT process and removes the contaminants from the sample that would cause problems with PCR and other downstream applications. 7. Centrifuge the tubes at 13,000 x g for 1 minute. 8. Avoiding the pellet, transfer the supernatant to a clean 2 ml Collection Tube (provided). Do not transfer more than 650 µl at this step. 9. Add 650 µl of Solution PM3 and 650 µl of Solution PM4. Vortex briefly to mix. Note: To prevent small RNA species (5S, trna and degraded RNA) from co-purifying with the mrna and rrna, use 650 µl of 70% ethanol instead of Solution PM4. For the purification of small RNAs, such as the microrna and sirna, transfer the lysate to a larger tube to accommodate a higher volume (2.6 ml) and add an additional 650 µl of 100% ethanol to the lysate. You will need to supply 100% ethanol for this step. What s happening: Solution PM3 contains the binding salts for total nucleic acid purification and Solution PM4 is 100% ethanol. These solutions set up the conditions for RNA and DNA binding to the Spin Filter. 9

10 10. Load 650 µl of supernatant onto a Spin Filter and centrifuge at 13,000 x g for 1 minute. Discard the flow through and repeat until all the supernatant has been loaded onto the Spin Filter. Note: A total of three loads for each sample processed is required and four loads if an additional volume of 100% ethanol is added for the microrna and sirna protocol. What s happening: The total nucleic acids are bound to the Spin Filter by passing it through the membrane using centrifugation. 11. Shake to mix Solution PM5. Add 650 µl of Solution PM5 and centrifuge at 13,000 x g for 1 minute. Note: Skip steps if you want to isolate both RNA and DNA. What s happening: Solution PM5 is an isopropanol containing wash buffer to remove salts from the membrane for optimal performance of the on-column DNase step. 12. Discard the flow through and centrifuge again at 13,000 x g for 1 minute to remove any residual wash. 13. Place the Spin Filter basket into a clean 2 ml Collection Tube (provided). 14. To the center of the Spin Filter, add 50 µl of DNase I Solution (prepared by mixing 46 µl of Solution PM6 and 4 µl of RTS DNase enzyme. See Important Notes Before Starting section). 15. Incubate at room temperature for 15 minutes. What s happening: Solution PM6 is a DNase digestion buffer. The DNase in the Solution PM6 soaks into the membrane and digests the genomic DNA on the column. 16. Add 400 µl Solution PM7 and centrifuge the column at 13,000 x g for 1 minute. What s happening: Solution PM7 inactivates the DNase enzyme and removes it from the column membrane along with digested DNA. 17. Discard the flow through and add 650 µl of Solution PM5 and centrifuge at 13,000 x g for 1 minute. 18. Discard the flow through and add 650 µl of Solution PM4 and centrifuge at 13,000 x g for 1 minute. What s happening: Solution PM5 and PM4 are isopropanol and ethanol containing wash buffers, respectively and are used to desalt the column before the elution step. 19. Discard the flow through and centrifuge again at 13,000 x g for 2 minutes to remove residual wash solution. What s happening: The final dry spin ensures all of the ethanol is cleared from the membrane so that the elution will be efficient. 20. Place the Spin Filter basket into a clean 2 ml Collection Tube (provided). 10

11 21. Add 100 µl of Solution PM8 (RNase-Free Water) to the center of the white filter membrane. Allow the water to sit on the membrane for at least 1 minute. Note: Eluting with 100 µl of Solution PM8 will maximize RNA yield. For more concentrated RNA, a minimum of 50 µl of Solution PM8 can be used. Do not use less than 50 µl of Solution PM Centrifuge at 13,000 x g for 1 minute. What s happening: RNA is solubilized from the Spin Filter membrane into Solution PM8 (RNase-Free Water) and is ready for use in enzymatic applications. 23. Discard the Spin Filter basket. The RNA is now ready for any downstream applications. The RNA in the tube can be stored at -80 C until use. Thank you for choosing the PowerMicrobiome RNA Isolation Kit! For further removal of genomic DNA post-rna extraction, we recommend the RTS DNase Kit, catalog # , which contains a DNase removal resin to generate DNAfree RNA without the use of heat or EDTA to inactivate enzymes. 11

12 Hints and Troubleshooting Guide RNA Degradation The optimal method for storing stool samples prior to RNA isolation is to freeze at -80 o C and to avoid multiple freeze/thaw cycles. The primary solid component of stool is dead bacteria which will contain a high level of degraded RNA. Co-isolation of degraded RNA along with intact RNA is expected. Removal of small degraded RNA is achieved by reducing the concentration of ethanol used at the binding step (step 9). Instead of Solution PM4, use 650 µl of 70% ethanol. If using stool samples preserved in RNALater, use only 0.1 gram of sample to avoid clogging the column. RNALater preserved samples will require additional DNase treatment post-purification to remove contaminating genomic DNA. The use of phenol:chloroform:isoamyl alcohol (25:24:1) buffered to ph is optional and may assist in protecting the integrity of the RNA during homogenization of the sample. Add 100 µl of phenol:chloroform:isoamyl alcohol to the Glass Bead Tube, 0.1 mm before adding the sample. For most solid samples, an organic phase and an aqueous phase may not be visible. However, for watery samples you may see a separation of phases. Always take the upper aqueous phase for the next step. Low Purity Expected purity readings for RNA are for the 260/280 ratio and >1.5 for the 260/230 ratio. If your sample has low purity, it may be related to the sample composition. If the 260/230 readings are low, increase the amount of Solution PM2 to 200 µl to remove more inhibitors. Alternatively, use less sample if the purity is not improved with the addition of more PM2. Genomic DNA contamination Depending on the quantity of bacterial cells in the starting sample, it is possible to still have residual genomic DNA in the RNA even after the on-column DNase digest. To completely remove the genomic DNA, a DNase treatment using the RTS DNase Kit Catalog # will ensure removal of DNA without the addition of EDTA or heat inactivation of the enzyme. 12

13 Contact Information Technical Support: Phone MO BIO Laboratories, Inc. Toll Free , or Fax: Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA Ordering Information: Direct: Phone MO BIO Laboratories, Inc. Toll Free , or Fax: Mail: MO BIO Laboratories, Inc, 2746 Loker Ave West, Carlsbad, CA For the distributor nearest you, visit our web site at 13

14 Products recommended for you For a complete list of products available from MO BIO Laboratories, Inc., visit Description Catalog No. Quantity DNase Max Kit preps PowerLyzer 24 Bench Top Bead-Based Homogenizer (110/220V) unit Vortex-Genie V 2 Vortex V-220 Vortex Adapter for 2 ml tubes V unit PowerVac Mini System unit (120 v) 1 unit (220 v) 1 unit + 20 adapters 14