Ribo-Zero Magnetic Kits*

Transcription

1 * Ribo-Zero Kit Catalog Number 6-Reactions Catalog Number 24-Reactions Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) MRZG126 MRZG12324 Ribo-Zero Magnetic Kit (Human/Mouse/Rat) MRZH116 MRZH11124 Ribo-Zero Magnetic Kit (Yeast) MRZY1306 MRZY1324 Ribo-Zero Magnetic Kit (Plant Leaf ) MRZPL116 MRZPL1224 Ribo-Zero Magnetic Kit (Seed and Root) MRZSR116 Not Available Ribo-Zero Magnetic Kit (Epidemiology) MRZE706 MRZE724 Ribo-Zero Magnetic Kit (Bacteria) MRZMB126 MRZB12424 Ribo-Zero Magnetic Kit (Gram-Negative Bacteria) MRZGN126 Not Available Ribo-Zero Magnetic Kit (Gram-Positive Bacteria) MRZGP126 Not Available Epicentre (an Illumina company) warrants that its products will retain full activity for 1 year from the date of receipt by the user if used and stored properly. For full warranty details, see Terms and Conditions available on the Epicentre website at * Covered by issued and/or pending patents. Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/epicentrebio), and Twitter (@EpicentreBio). Lit. # 410 8/ EPILIT410 Rev. B

2 Table of Contents Page Choosing the Optimal Ribo-Zero Kit...3 Kit Contents...4 Preparation...6 Ribo-Zero Protocol...7 Workflow Overview...7 Quick Protocol for Experienced Users...8 Step 1: Wash the Magnetic Beads...9 Step 2: Treat the Sample With the rrna Removal Solution Step 3. Remove the rrna Step 4. Purify the Ribo-Zero Treated RNA Quantify the Yield and Assess the Quality of the Ribo-Zero Treated RNA Troubleshooting Guide

3 Choosing The Optimal Ribo-Zero Kit Ribo-Zero Species Compatibility and rrna removal Ribo-Zero kits can be used successfully with a broad range of species. The efficiency of rrna removal depends on the factors such as species, sample quality, and sequence homology between rrna samples and Ribo-Zero Removal Solutions. Don t See your Organism? Please visit our RNAMatchMaker tool to determine the best Ribo-Zero kit for your sample at: Ribo-Zero Kit Depletion Guide Kit Name Ribo-Zero Gold (Human/Mouse/Rat) Ribo-Zero (Human/Mouse/Rat) Ribo-Zero (Bacteria) Ribo-Zero (Gram-Negative Bacteria) Ribo-Zero (Gram-Positive Bacteria) RNA Sample Human and other animal species; FFPE RNA Human and other animal species and FFPE RNA Bacteria (mixed populations of gram-negative and gram-positive bacteria) Gram-negative bacteria Gram-positive bacteria Removes Euk. Cytoplasmic rrna Removes Mitochondrial rrna Removes Bacterial rrna Removes Chloroplast rrna Ribo-Zero (Plant Leaf) Plant leaf tissue Ribo-Zero (Plant Seed/Root) Plant seed or root tissue Ribo-Zero (Yeast) Yeast Ribo-Zero (Epidemiology) Human microbiome and other bacteria-infected human, mouse, rat samples techhelp@epicentre.com (800)

4 Kit Contents The Ribo-Zero Magnetic Kits are composed of two parts: Ribo-Zero rrna Removal Reagents Magnetic Core Kit Ribo-Zero Kit Selection Guide Kit Name Size Catalog Number (Included Kit Sub-Components) *Not Available for Individual Sale *Part 1 *Part 2 Ribo-Zero rrna Removal Reagents (Store 65 C to 80 C) Magnetic Core Kit (Store +2 C to +8 C) Ribo-Zero Gold (Human/Mouse/Rat) Ribo-Zero (Human/Mouse/Rat) Ribo-Zero (Bacteria) Ribo-Zero (Gram-Negative Bacteria) Ribo-Zero (Gram-Positive Bacteria) Ribo-Zero (Plant Leaf) 6 MRZG126 RZHM11106 MRZ116C 24 MRZG12324 RZG1224 MRZ11124C 6 MRZH116 RZH1046 MRZ116C 24 MRZH11124 RZH MRZ11124C 6 MRZMB126 RZMB11086 MRZ116C 24 MRZB12424 RZMB12324 MRZ11124C 6 MRZGN126 RZMB1056 MRZ116C 6 MRZGP126 RZPB10106 MRZ116C 6 MRZPL116 RZPL11016 MRZ116C 24 MRZPL1224 RZPL1224 MRZ11124C Ribo-Zero (Plant Seed/Root) 6 MRZSR116 RZSR11036 MRZ116C Ribo-Zero (Yeast) Ribo-Zero (Epidemiology) 6 MRZY1306 RZY1306 MRZ116C 24 MRZY1324 RZY1324 MRZ11124C 6 MRZE706 RZE1206 MRZ116C 24 MRZE724 RZE1224 MRZ11124C 4

5 Ribo-Zero rrna Removal Reagents; Store at 65 C to 80 C Volume Part 1 Kit Component 6-rxn kits 24-rxn kit RiboGuard RNase Inhibitor 10 μl 30 μl Ribo-Zero rrna Removal Solution 75 μl 27 μl Ribo-Zero rrna Reaction Buffer 50 μl 110 μl Glycogen (10 mg/ml) 20 μl 60 μl Sodium Acetate (3 M) 150 μl 500 μl RNase-Free Water 2 x 1 ml 3 x 1.5 ml Cap Color Blue Clear Storage 65 C to 80 C Magnetic Core Kit; Store at +2 C to +8 C Do Not Freeze! Part 2 Kit Component 6-rxn kits Volume 24-rxn kit Cap Color Magnetic Beads 1.4 ml 5.4 ml Clear Magnetic Bead Resuspension Solution 500 μl 2 ml Yellow/ Clear RNase-Free Water 3 ml 2 x 5.5 ml Clear Storage +2 C to +8 C (Do not Freeze!) Additional Required Reagents and Equipment for the Ribo-Zero Procedure: Magnetic rack or stand for 1.5 ml tubes (e.g., Cat. No. LS001, MS002, MS003 from Bangs Laboratories, Inc. or Cat. No D, DynaMag -2 magnet from Life Technologies) or magnetic plate (Cat. No , DynaMag -96 Side from Life Technologies) Optional: Magnetic rack or stand for 15 ml tubes for batch washing the Magnetic Beads in Step 1.B (e.g., PureProteome Magnetic Stand, EMD Millipore) 1.5 ml microcentrifuge 1.5 ml microcentrifuge tubes (RNase-free) 0.2 ml or 0.5 ml microcentrifuge tubes (RNase-free) Thermocycler, water bath, heating block, or other temperature control device RNA purification kits such as Agencourt RNAClean XP kit (Beckman Coulter), or RNA Clean & Concentrator -5 column (Zymo Research), RNeasy MinElute Column (Qiagen), or ice-cold 100% ethanol for ethanol precipitation Vortex Mixer For Ethanol Precipitation Purification 70% (v/v) and 100% ice-cold Ethanol 3 M Sodium Acetate 10 mg/ml Glycogen techhelp@epicentre.com (800)

6 Preparation Read this protocol closely before performing the Ribo-Zero reaction. Important! DNA-free RNA The RNA samples must be free of contaminating DNA before beginning the Ribo-Zero procedure. Treat the sample with DNase and then purify the DNase-treated RNA. We recommend using Baseline-ZERO DNase (Epicentre; catalog numbers DB0711K or DB0715K). Contaminating DNA may lead to inaccurate RNA quantitation, interfere with rrna removal and negatively affect RNA-Seq library prep and sequencing. The RNA sample must be free of salts (e.g., Mg 2+ or guanidinium salts) and organics (e.g. phenol and ethanol). Important! RNA Quantification It is important to quantify the amount of total RNA in the sample as accurately as possible (e.g. using a fluorescent assay). Accurate RNA quantitation is necessary in order to use the correct amount of Ribo-Zero rrna Removal Solution in Step 2, Table 1 or Table 2 to determine the maximum volume in which the total RNA sample can be dissolved prior to performing the Ribo-Zero procedure. We recommend use of water or TE buffer for sample dilution. Do not exceed recommended input amounts of total RNA (5 μg total RNA for most Ribo-Zero Kits or 2.5 ug of total RNA for the Ribo-Zero (Epidemiology) Kit.); exceeding these limits will reduce rrna removal efficiency. Important! Magnetic Core Kit & Temperature Contents of the Magnetic Core Kit (magnetic beads) should never be frozen or put on ice. Freezing the Magnetic Beads will decrease rrna removal and Ribo-Zero kit performance. Beads should be used at room temperature when performing Ribo-Zero rrna depletion, and stored at +2 C to +8 C when not in use. FFPE-RNA samples RNA extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue can be used with Ribo-Zero kits. However, the quality of the FFPE RNA is highly variable due to the tissue-fixation procedure, age of sample, storage conditions, fixation reversal process, etc. Therefore, we cannot guarantee success with every FFPE RNA sample. Automation and Ribo-Zero Epicentre does not provide support for any liquid handling robotic platforms or protocols. Please contact the manufacture of the liquid handling instrument for hardware, software, protocol, and technical support. Do not use conductive tips when using an automated liquid handler with Ribo-Zero magnetic beads. Performance Specifications and Quality Control Ribosomal RNA (rrna) removal is tested by qrt-pcr using total RNA from kit-specific model organisms. Depletion is assessed before and after a Ribo-Zero Magnetic Kit reaction a Ribo-Zero Magnetic Kit reaction removes: >99% of the large cytoplasmic rrna and where applicable, the large mitochondrial and chloroplast rrna. >85% to 95% of the small (e.g., 5S) rrnas. Results may vary, especially with 5S rrna removal, depending on the species, quality of individual sample, and sequence homology between rrna samples and Ribo-Zero Removal Solutions. We recommend verifying sample/ribo-zero kit rrna sequence homology using the RNAMatchMaker tool (see: 6

7 Ribo-Zero Protocol Epicentre Ribo-Zero Workflow (4-Steps) Steps) Step 1. Wash the magnetic beads. Step 2. Treat sample with rrna Removal Solution. Magnetic beads in storage buffer Pellet and wash magnetic beads (x2) Magnet Non-rRNA rrna Non-rRNA Removal Probes Mix: Total RNA (DNase-treated sample) rrna Removal Solution (Probes) Incubate Resuspend magnetic beads (+ optional RiboGuard RNase Inhibitor) (Treated Sample) rrna Removal Probes Hybridize to rrna Step 3. Remove rrna. Mix immediately by pipetting. Add treated (Hybridized) sample to washed magnetic beads Magnetic beads bind to rrna removal probes Pellet magnetic beads (including bound rrna and excess unbound probes) Magnet Magnet Recover rrna-depleted Sample Non-rRNA Step 4. Purify rrna-depleted sample. (Several clean-up options available) rrna-depleted sample ready for library prep! Legend: Magnetic Beads Probe Non-rRNA rrna (800)

8 Quick Protocol for Ribo-Zero Kit For experienced users only! The detailed procedure begins at Step 1 below. Step Procedure Pages 1. Prepare Magnetic Beads Add 225 µl Magnetic Beads to RNase-free tube Remove supernatant, place in magnetic stand for 1 minute at RT Wash with 225 µl RNase-Free Water Place in magnetic stand for 1 min, repeat wash step Remove any remaining water Resuspend in 65 µl Resuspension Solution Optional: Add 1 µl RiboGuard RNase Inhibitor Store at room temperature Treat sample with rrna Removal Solution Mix in 40 µl total volume: 1-5 µg total RNA (500 ng to 2.5 ug using the Ribo-Zero (Epidemiology) Kit) 8 µl -10 µl rrna Removal Solution 4 µl Reaction Buffer Incubate C, then 5 RT Remove rrna Vortex previously prepared Magnetic Beads Add RNA mixture to the washed beads, mix well by vortex Vortex for at least 10 seconds Incubate 5 RT, vortex Incubate 5 50 C Place in magnetic stand for 1 min, transfer supernatant (rrna-depleted sample) to RNase-free tube Purify rrna-depleted sample Ethanol precipitation or alternative method

9 Step 1. Wash the Magnetic Beads Magnetic Beads must be washed prior to using the Ribo-Zero kits. The Magnetic Beads can be washed in single-sample aliquots (Step 1A.) or by batch washing (Step 1B.) It is important to use beads at room temperature. Important! Do not freeze or place the Magnetic Beads on ice! Damaged (frozen) beads will impact rrna removal and kit performance! 1A. Individual Washing Procedure Required for Individual washing (provided by the user): RNase-free microfuge tubes with cap Vortex mixer Magnetic stand or rack 1A.1 Remove Magnetic Core Kit components from +2 C to +8 C storage. 1A.2 Allow the Magnetic Core Kit components to equilibrate to room temperature. Do not place Magnetic Core Kit or Beads on ice! 1A.3 Vigorously vortex the Magnetic Beads to homogeneity. 1A.4 For each reaction, pipette 225 µl of Magnetic Beads slowly to avoid air bubbles into an RNase-Free tube. Store unused beads at +2 C to +8 C. Do not place Magnetic Beads on ice! 1A.5 Place each tube, with cap open, on a magnetic stand for at least 1 minute (until the solution becomes clear). 1A.6 With the tube still on the magnetic stand, remove and discard the supernatant. Caution! The supernatant contains 0.1% Sodium Azide. 1A.7 Remove each tube from the magnetic stand and add 225 µl of RNase-Free Water; Fully re-suspend Magnetic Beads by vortexing. 1A.8 Repeat steps 1A.5-1A.7 for a total of 2 water washes. 1A.9 Place each tube, with cap open, on a magnetic stand for 1 minute. Remove and discard remaining water. 1A.10 Remove each tube from the magnetic stand and add 65 µl of Magnetic Bead Resuspension Solution and resuspend the beads by vortex. Optional: Add 1 µl RiboGuard RNase Inhibitor to each tube of resuspended Magnetic Beads and pipette mix times. Avoid creating air bubbles). 1A.11 Store the washed Magnetic Beads at room temperature until used in Step 3. 1B. Batch Washing Procedure Required for batch washing procedure (provided by the user): RNase-free 1.5 ml tubes Optional: 15 ml tube Vortex mixer Magnetic stand or rack For each Ribo-Zero reaction, 225 µl of the Magnetic Beads is required. Up to 1,350 µl of Magnetic Beads (sufficient for 6 Ribo-Zero reactions) can be washed in a 1.5 ml tube. Use a 15 ml tube if batch washing more than 1,350 µl of Beads. 1B.1 Remove the Magnetic Core Kit components from +2 C to +8 C storage and allow them to equilibrate to room temperature. Do not place Magnetic Core Kit or Beads on ice! 1B. 2 Vigorously vortex the Magnetic Beads to homogeneity. 1B. 3 Pipet the Magnetic Bead suspension slowly to avoid air bubbles and to ensure pipetting of the correct volume. 1B. 4 Place the tube containing the Magnetic Beads on the magnetic stand for at least 1 minute until the solution appears clear. 1B. 5 With the tube still on the stand, remove and discard the supernatant. Caution: The supernatant contains 0.1% sodium azide. techhelp@epicentre.com (800)

10 1B. 6 Remove the tube from the stand and add an equal volume of RNase-Free Water. Vigorously vortex the Magnetic Beads. 1B. 7 Repeat steps 1B.4, 1B.5 and 1B.6 (i.e. wash the beads a total of 2 times with RNase-Free Water). 1B. 8 After the second water wash, place tube in a magnetic stand for 1 minute, then remove and discard the water. 1B. 9 Remove the tube from the magnetic stand. Add a volume of Magnetic Bead Resuspension Solution equal to the number of reactions x 60 µl (e.g., for 6 reactions, add 6 x 60 µl = 360 µl Magnetic Bead Resuspension Solution). Mix well by vigorous vortexing. Note: The volumes of the beads and Resuspension Solution are additive. Although the washed beads are resuspended in 60 µl per reaction, each reaction uses 65 µl of resuspended beads. 1B. 10 Aliquot 65 µl of the washed and resuspended Magnetic Beads into new 1.5-ml RNase-free microcentrifuge tubes corresponding to the number of Ribo-Zero reactions. 1B. 11 Optional: Add 1 µl of RiboGuard RNase Inhibitor to each tube of resuspended Magnetic Beads, and mix briefly by vortexing. 1B. 12 Store the washed Magnetic Beads at room temperature until required in Step 3. Return the remaining Magnetic Core Kit to +2 C to +8 C. Step 2. Treat Sample with rrna Removal Solution Important! In this step, the rrna Removal Solution (probes) hybridizes to the ribosomal RNA in the sample. RNA samples must be DNase-treated and purified prior to treatment with rrna Removal Solution. Required in Step 2 Kit Component Ribo-Zero Reaction Buffer Ribo-Zero rrna Removal Solution RNase-Free Water Cap Color Blue Clear Required for each reaction (provided by user): 0.2-ml or 0.5-ml microcentrifuge tube (RNase-free) Important! The maximum volume of the RNA sample and the volume of the Ribo-Zero rrna Removal Solution used per reaction is dependent on the amount of total RNA in the sample. Use Table 1 for all Ribo-Zero Kits EXCEPT the Ribo-Zero (Epidemiology) Kit. Use Table 2 if working with the Ribo-Zero (Epidemiology) Kit. Table 1. Volumes of Ribo-Zero rrna Removal Solution for all Ribo-Zero kits EXCEPT the Ribo-Zero (Epidemiology) Kit. See Table 2 below if using the Ribo-Zero (Epidemiology) Kit. Amount of Input Total RNA Maximum volume of total RNA that can be added to each reaction Volume of Ribo-Zero rrna Removal Solution used per reaction μg 28 μl 8 μl >2.5 μg - 5 μg 26 μl 10 μl Table 2. Ribo-Zero (Epidemiology) rrna Removal Solution. Use this table if working with the Ribo-Zero (Epidemiology) Kit. Amount of Input Total RNA Maximum volume of total RNA that can be added to each reaction Volume of Ribo-Zero rrna Removal Solution used per reaction 500 ng μg 28 μl 8 μl >1.25 ug μg 26 μl 10 μl 10

11 2.1 Remove Ribo-Zero rrna Removal Probes components from 65 C to 80 C storage and allow them to equilibrate to room temperature For each sample, add the following reagents in a 0.2 ml or 0.5 ml RNase-free microcentrifuge tube. Combine in the order given: x μl RNase-Free Water 4 μl Ribo-Zero Reaction Buffer y μl RNA Sample (see Table 1 or Table 2) 8-10 μl Ribo-Zero rrna Removal Solution (see Table 1 or Table 2) 40 µl Total Volume 2. 3 Fully mix by pipetting times; incubate at 68 C for 10 minutes. Return the remaining Ribo-Zero rrna Removal Solution and Ribo-Zero Reaction Buffer to 65 C to 80 C Remove tubes from heat and quick spin to collect any condensation Incubate tubes at room temperature for 5 minutes. Step 3. Remove rrna This is the most important Step in the Ribo-Zero procedure. This step combines probe-hybridized samples from Step 2.5 to the washed room temperature Magnetic Beads from Step 1. Important! The washed Magnetic Beads from Step 1 must be at room temperature for use in this step. Order of this addition is critical and may impact rrna removal efficiency if done incorrectly! 3.1 Transfer probe-hybridized RNA sample to the washed room temperature Magnetic Beads. 40 µl Probe-hybridized RNA sample 65 μl Washed room temperature Magnetic Beads 105 µl Total Volume Without changing the pipet tip, immediately and thoroughly mix the contents of the tube by pipetting times. Set the tube aside at room temperature and repeat Step 3.1 for each sample Cap the tubes and vortex at high speed for a minimum of 10 seconds. Avoid vortexing beads/sample into tube cap. Perform this process for each sample Incubate tubes at room temperature for 5 minutes Incubate tubes at 50 C for 5 minutes Remove tubes from heat and immediately place them on a magnetic stand for at least 1 minute until the solution appears clear Carefully remove supernatant (85-90 µl) containing the rrna-depleted sample and transfer to a labeled 1.5-ml RNase-free microcentrifuge tube. Important! The supernatant contains rrna-depleted RNA! Step 3. Remove rrna. Mix immediately by pipetting. Add treated (Hybridized) sample to washed magnetic beads Tip: Remove all Magnetic Beads from the sample. The Magnetic Beads have the unwanted rrna and Ribo-Zero probes bound to them. If any Magnetic Beads are still visible in the supernatant, place the collected supernatant onto the magnetic stand for 1 minute. Remove the supernatant containing the rrna-depleted RNA and transfer to a new 1.5 ml RNase-free microcentrifuge tube Place the supernatant (rrna-depleted sample) on ice and proceed to Step 4 (Purify the Ribo-Zero treated RNA). Alternatively, the supernatant may be stored at 20 C overnight or 65 C to 80 C long term storage. techhelp@epicentre.com (800)

12 Step 4. Purify the Ribo-Zero treated RNA Ribo-Zero rrna-depleted samples require purification prior to downstream library prep. This purification step removes any remaining salts/buffers, and also allows the rrna-depleted RNA samples to be concentrated. Samples can be purified by one of these methods: Option 1: Ethanol precipitation Option 2: Agencourt RNAClean XP Beads (BeckmanCoulter) Option 3: Modified RNeasy MinElute cleanup Kit (Qiagen) Option 4: RNA Clean & Concentrator columns (Zymo Research) Ethanol precipitation and the modified RNeasy method provide optimal recovery of both large and small RNAs. The presence of a high proportion of small RNAs in the Ribo-Zero treated samples should not be interpreted as degraded RNA. Ethanol precipitation can be challenging for inexperienced users due to the extremely small RNA pellet recovered after centrifugation. Ethanol precipitation recovers small RNAs such as mirna and trna along with the mrnas and large non-coding RNAs. The Agencourt RNAClean XP procedure presented here will not quantitatively recover small RNAs. The RNA Clean & Concentrator columns can be used to recover both large and small RNAs or just the large (>200 b) RNAs. Purification Option 1: Ethanol Precipitation of the rrna-depleted Sample Additionally required reagents and materials (provided by user): Ice-cold 70% (v/v) and 100% (absolute) Ethanol 3 M Sodium Acetate 10 mg/ml Glycogen 1. Adjust the volume of each sample to 180 μl using RNase-Free Water. 2. Add 18 μl of 3 M Sodium Acetate to each tube. 3. Add 2 μl of Glycogen (10 mg/ml) to each tube and mix by gentle vortexing. 4. Add three volumes (600 μl) of ice-cold 100% ethanol to each tube and mix thoroughly by gentle vortexing. 5. Place the tubes at 20 C for at least 1 hour. 6. Centrifuge the tubes at >10,000 x g in a microcentrifuge for 30 minutes. Carefully remove and discard the supernatant. 7. Wash the pellet with ice-cold 70% (v/v) ethanol and centrifuge at >10,000 x g for 5 minutes. Carefully remove and discard the supernatant. 8. Repeat Step 7 (above); for total of two 70% (v/v) ethanol washes. 9. Centrifuge briefly to collect any residual supernatant. Carefully remove and discard the supernatant and allow the pellet to air dry at room temperature for 5 minutes. 10. Dissolve the pellet in 10 μl of RNase-Free Water or buffer. 11. Place on ice for immediate use or store 20 C overnight or at 65 C or 80 C for long term storage. 12

13 Purification Option 2: Agencourt RNAClean XP Kit Additionally required reagents and material (provided by user): Agencourt RNAClean XP Kit (Cat. No. A63987, Beckman Coulter) Freshly prepared 80% (v/v) Ethanol Magnetic stand or rack 1. Place samples at room temperature for 2 minutes. 2. Vortex the AMPure RNAClean XP Beads until homogeneous and free from the bottom of the bottle. 3. Add 160 μl of the mixed AMPure XP Beads (1.6x) to each tube containing µl of rrna-depleted RNA from Step 3 (Remove rrna). 4. Mix thoroughly by gently pipetting the entire volume 10 times. 5. Incubate the tube(s) at room temperature for 15 minutes. 6. Place the tube(s) on the magnetic stand at room temperature for at least 5 minutes (until the liquid appears clear). 7. Remove and discard the supernatant from each tube. Do not disturb the beads. 8. With the tube(s) still on the magnetic stand, add 200 μl of freshly prepared 80% (v/v) ethanol to each tube, without disturbing the beads. 9. Incubate at room temperature for at least 30 seconds while still on the magnetic stand. Then remove and discard all of the supernatant from each tube. Do not disturb the beads. 10. Repeat Steps 8 and 9 (total of two 80% (v/v) ethanol washes). 11. Allow the tubes to air dry on the magnetic stand at room temperature for 15 minutes. 12. Add 11 µl of RNase-Free water to each tube and thoroughly resuspend the beads by gently pipetting 10 times. 13. Incubate the tubes at room temperature for 2 minutes. 14. Place the tubes back onto the magnetic stand at room temperature for at least 5 minutes (until the liquid appears clear). 15. Transfer the clear supernatant (contains the rrna-depleted RNA!) from each tube to an appropriate collection tube, always leaving at least 1 μl of the supernatant behind to avoid carryover of magnetic particles. 16. Store on ice for immediate use or store 20 C overnight or at 65 C or 80 C for long term storage. Purification Option 3: Modified RNeasy MinElute Cleanup Kit Additionally required reagents and material (provided by user): RNeasy MinElute Cleanup Kit (Cat No , Qiagen) 96% (v/v) - 100% Ethanol 1. Adjust the sample to a volume of 100 μl with RNase-Free Water. Add 350 μl of Buffer RLT, and mix well. 2. Add 550 μl of 96%-100% (v/v) ethanol to the RNA, and mix well by pipetting. Do not centrifuge. 3. Transfer half of the sample (~500 µl) to an RNeasy MinElute spin column placed in a 2-ml collection tube (supplied in the Qiagen kit). Close the lid gently, and centrifuge for 15 seconds at 8,000 x g (~10,000 rpm). Discard the flowthrough. Reuse the collection tube from Step 5 (below). 4. Transfer the remaining sample and repeat the centrifugation. Discard the flow-through and collection tube. 5. Place the RNeasy MinElute spin column in a new 2-ml collection tube (supplied in the Qiagen kit). Add 500 μl Buffer RPE to the spin column. Close the lid gently, and centrifuge for 15 seconds at 8,000 x g (~10,000 rpm) to wash the spin-column membrane. Discard the flow-through. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use. 6. Add 500 μl of 80% (v/v) ethanol to the RNeasy MinElute spin column. Close the lid gently, and centrifuge for 2 minutes at 8,000 x g (~10,000 rpm) to wash the spin-column membrane. After centrifugation, carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur. 7. Discard the flow-through and collection tube. Place the RNeasy MinElute spin column in a new 2-ml collection tube that is supplied in the Qiagen kit. techhelp@epicentre.com (800)

14 8. Open the lid of the spin column, and centrifuge at full speed for 5 minutes. Discard the flow-through and collection tube. Note: To avoid damage to the spin-column lids, place the spin columns into the centrifuge with at least one empty position between columns. Orient the lids so that they point in a direction opposite to the rotation of the rotor (i.e., if the rotor rotates clockwise, orient the lids counterclockwise). Note: It is important to dry the spin-column membrane since residual ethanol may interfere with downstream applications. Centrifugation with the lids open ensures that no ethanol is carried over during RNA elution. 9. Place the RNeasy MinElute spin column in a new 1.5-ml collection tube (supplied in the Qiagen kit). Add 12 μl of RNase-Free Water directly to the center of the spin-column membrane. Close the lid gently, and centrifuge for 1 minute at full speed to elute the RNA. Recovery is usually 10 μl. 10. Store on ice for immediate use or store 20 C overnight or at 65 C or 80 C for long term storage. Purification Option 4: RNA Clean & Concentrator 5 columns (Zymo Research) Additionally required reagents and material (provided by user): RNA Clean & Concentrator (Cat No. R1015, R1016, Zymo) 96% (v/v) - 100% Ethanol 1. Follow the manufacturer s procedure for either purification of RNA >17 nts or for purification of RNA >200 nts. 2. Store on ice for immediate use or store 20 C overnight or at 65 C or 80 C for long term storage. Quantify the Yield and Assess the Quality of the rrna-depleted Samples The yield of rrna-depleted RNA is dependent on the amount of input total RNA, rrna content of the sample, and the method used to purify the Ribo-Zero treated RNA. Typically, <8% of the amount of input RNA is recovered (e.g. 1 µg total RNA input will typically yield <80 ng of rrna-depleted RNA). We recommend using the 2100 Bioanalyzer to quantify the yield of Ribo-Zero treated RNA. Other methods such as standard absorbance (e.g. NanoDrop spectrophotometer) or fluorometry (e.g. Qubit Flourometer) should be used with caution. The NanoDrop and Qubit may not give accurate quantification if the original total RNA sample had a low endogenous mrna content or the post-ribo-zero purification method used in Step 4 removed the trna and other small RNAs. When assessing the quality of the Ribo-Zero treated RNA using an Agilent 2100 Bioanalyzer, use the Agilent RNA6000 PicoChip and load 1 μl of the Ribo-Zero treated RNA. The Agilent RNA NanoChip do not provide sufficient sensitivity. When purifying the Ribo-Zero treated RNA by ethanol precipitation, the modified RNeasy column method or the RNA Clean & Concentrator column method, small RNAs such as mirna and trna are recovered along with the mrnas and large noncoding RNAs. Therefore, the presence of a high proportion of small RNA in the Ribo-Zero treated sample should not be interpreted as degradation of the RNA. 14

15 A B C Figure 1. Typical Before/After Ribo-Zero rrna-depletion Traces run on the Agilent Bioanalyzer using the RNA6000 Pico Chip. A. Universal Human Reference Total RNA diluted to 25 ng/μl (1μl loaded). B & C. 1 μg of Universal Human Reference Total RNA was treated with Ribo-Zero Gold (Human/Mouse/Rat) to remove rrna and purified using AMPure beads. Please notice the scale in the traces. Results are similar with other organisms. For more example of Bioanalyzer profiles, see the Ribo-Zero FAQs on the Epicentre website at (Optional) For further validation We highly recommend creating qrt-pcr primers to your species rrna along with a housekeeping gene to verify that the rrna has been completely removed from your sample. techhelp@epicentre.com (800)

16 Troubleshooting Guide Observation / Cause Recommendation(s) Incomplete removal of rrna Species compatibility Too much RNA used DNA contamination Insufficient rrna Removal Solution used The Magnetic Beads were not at room temperature when used The Magnetic Beads were frozen Incorrect addition of RNA sample to magnetic beads Insufficient sample mixing Badly degraded RNA sample Incomplete magnetic bead removal The Ribo-Zero kits were developed using model organisms. Results may vary, especially with 5S rrna removal, depending on your species. Confirm the compatibility of your species with the Ribo-Zero kit using the RNA MatchMaker on the Epicentre website. The Ribo-Zero kits use 1 to 5 µg of total RNA (500 ng to 2.5 µg total RNA for the Ribo-Zero Epidemiology Kit). Do not exceed the maximum amount of RNA. We recommend quantifying the input total RNA using the Qubit. The RNA concentration reported by the Bioanlyzer or NanoDrop may not be accurate. RNA samples need to be DNase treated previous to Ribo-Zero or the probes may bind to the DNA reducing rrna removal. The amount of the rrna Removal Solution to use is dependent on the amount of total RNA input. Accurately quantify the amount of RNA and then consult Table 1 or Table 2 in Step 2 to determine the correct volume of the rrna Removal Solution. The Magnetic Beads provided in the Magnetic Core Kit must be at room temperature prior to washing (Step 1). Once washed, the beads must be kept at room temperature for use in rrna removal in Step 3. The Magnetic Beads will not work if frozen. Confirm that the beads were not frozen when they arrived in the lab and that they were stored in the refrigerator at +2 C to +8 C. Do Not Freeze the magnetic beads! Be sure to add the RNA sample to the washed, room temperature Magnetic Beads in Step 3. DO NOT add the Magnetic Beads to the RNA sample. Follow the Ribo-Zero procedure closely. The procedure requires frequent and complete mixing for successful rrna removal. The Ribo-Zero kits will remove rrna from partially degraded samples but severely degraded samples may not be removed efficiently due to inability of probes to hybridize. Failure to completely remove the Magnetic Beads will cause carryover of the Ribo-Zero probes and rrna. Place the tubes on a magnetic stand for 5 minutes and then carefully transfer the supernatant containing the rrna-depleted RNA to a new RNase-free tube. 16

17 Low recovery rrna content of the sample Species compatibility Method of purification Quantifying the recovered RNA Some samples contain a higher proportion of rrnas or lower proportions of mrna and large non-coding RNAs than others. The Ribo-Zero kits were developed using model organisms. rrna removal and RNA recovery may vary depending on your species or cell type. Small RNAs (e.g., trna, mirna, etc.) make up a high proportion of the Ribo- Zero treated RNA. If the method used to purify the treated RNA removes small RNA (e.g. AMPure bead purification), the recovery may appear to be lower than expected. Please read Step 4 for sample cleanup options. Different methods used to quantify the recovered RNA will give different results. We recommend using the 2100 Bioanalyzer for most accurate quantification of the recovered RNA. Degraded RNA after Ribo-Zero RNA looks degraded post Ribo-Zero treatment Small RNAs such as mirna and trna, etc. are recovered along with the mrnas and large noncoding RNAs. Therefore, the presence of a high proportion of small RNA (less than 200 nt by Bioanalyzer) in the treated sample should not be interpreted as degradation of the RNA. Representative 2100 Bioanalyzer (Agilent) profile of Ribo-Zero treated RNAs. Magnetic beads don t resuspend Magnetic Beads froze during storage When adding RNA sample to beads they clump Be sure to store the Magnetic Beads at +2 to +8 C. Never freeze the magnetic beads. The beads may form clumps after addition of the probe: RNA hybridized sample. The probes contains multiple biotins that may be bound by multiple magnetic bead particles and crosslink the beads into clumps. Be sure to vortex the beads vigorously for a minimum of 10 seconds during Step 3. rrna reads in sequencing data Insufficient sample mixing Incomplete Magnetic Bead removal Follow the Ribo-Zero procedure closely. The procedure requires frequent and complete mixing for successful rrna removal. Failure to completely remove the Magnetic Beads will cause carryover of the Ribo-Zero probes and rrna. Place the tubes on a magnetic stand for 5 minutes and then carefully transfer the supernatant containing the rrna-depleted RNA to a new RNase-free tube. Also see Incomplete Removal of rrna section Ribo-Zero, Baseline-ZERO, and ScriptSeq are trademarks of Epicentre, Madison, Wisconsin. NanoDrop is a registered trademark of NanoDrop Technologies Inc., Wilmington, Delaware. RiboGreen is a registered trademark of Molecular Research Center Inc., Cincinnati, Ohio. RNAClean is a trademark of and Agencourt is a registered trademark of BeckmanCoulter, Brea, CA. RNeasy is a registered trademark and MinElute is a trademark of Qiagen Inc. Clean & Concentrator is a trademark of Zymo Research Corp., Irvine, CA Visit our technical blog: epicentral.blogspot.com techhelp@epicentre.com (800)