Ribo-Zero rrna Removal Kit * (Human/Mouse/Rat)

Transcription

1 Ribo-Zero rrna Removal Kit * (Human/Mouse/Rat) Cat. No. RZH Reactions (Contains 1 box of Cat. No. RZH1046 and 1 box of Cat. No. RZC1046) * Patent Pending EPICENTRE Biotechnologies Lit. # /2010 1

2 Table of Contents 1. Kit Contents Storage Additional.Required.Reagents.and.Equipment Performance.Specifications.and.Quality.Control Introduction Advantages.of.the.Ribo-Zero.rRNA.Removal.Kit.(Human/Mouse/Rat)... Compared.to.other.rRNA.Removal.Methods Summary.of.the.Ribo-Zero.rRNA.Removal.Process Preparation Maintaining.an.RNase-Free.Environment Input.Total.RNA Ribo-Zero.rRNA.Removal.Solution.(Human/Mouse/Rat) Ribo-Zero.Microspheres Purification.of.the.rRNA-Depleted.Sample Yield.of.rRNA-Depleted.RNA Familiarize.Yourself.with.the.Ribo-Zero.rRNA.Removal.Kit... (Human/Mouse/Rat).and.Procedure Control.Reaction Ribo-Zero rrna Removal kit (Human/Mouse/Rat) Procedure Prepare.the.Ribo-Zero.Microspheres Treat.the.Total.RNA.Sample.with.Ribo-Zero.rRNA.Removal.Solution Microsphere.Reaction.and.rRNA.Removal Purify.the.rRNA-Depleted.Sample Ethanol.Precipitation.of.the.rRNA-Depleted.Sample Column.Purification.of.the.rRNA-Depleted.Sample Quantifying the Yield and Assessing the Quality of the rrna-depleted Sample Related Products EPICENTRE Biotechnologies

3 1. Kit Contents The.kit.components.are.supplied.in.tubes.with.colored.caps.for.easier.identification. Each.kit.contains.one.box.of.Cat..No..RZH1046.and.one.box.of.Cat..No..RZC1046. Ribo-Zero rrna Removal Kit (Human/Mouse/Rat) Cat. No. RZH1046 Component Name Tube Label Volume.. Blue-Cap.Tubes.. RiboGuard.RNase.Inhibitor.(100.U/μl). RiboGuard.RNase.Inhibitor. 10.μl Ribo-Zero.rRNA.Removal.Solution. rrna.removal.solution.(h/m/r). 75.μl...(Human/Mouse/Rat). Ribo-Zero.Reaction.Buffer. Ribo-Zero.Reaction.Buffer. 50.μl.. Colorless-Cap.Tubes.. Glycogen.(10.mg/ml). Glycogen. 20.μl Sodium.Acetate.(3.M). Sodium.Acetate. 150.μl RNase-Free.Water. RNase-Free.Water. 2.x.1.ml Storage:.Store.this.kit.box.and.its.contents.at. 70 C to 80 C. Ribo-Zero Core Kit Cat. No. RZC1046 Component Name Tube Label. Volume.. Green-Cap.Tubes... Ribo-Zero.Microsphere.Wash.Solution. Microsphere.Wash.Solution. 1.ml.. Red-Cap.Tubes.. Ribo-Zero.Microsphere.Resuspension. Resuspension.Solution. 500.μl...Solution... Colorless-Cap.Tubes.. Ribo-Zero.Microspheres. Microspheres. 500.μl...(contains.0.1%.sodium.azide) Microsphere.Wash.Tube.(2.ml). Microsphere.Wash.Tube. 6.tubes Microsphere.Removal.Filter.Unit.(sterile). Microsphere.Removal.Unit. 6.filter.units Storage:.Store.this.kit.box.and.its.contents.at.4 C.(Do Not Freeze!). Additional Required Reagents and Equipment: 0.2-ml.or.0.5-ml.microcentrifuge.tubes.(RNase-Free) Thermocycler.or.other.temperature.control.device.for.0.2-ml.or.0.5-ml.tubes Water.bath,.heating.block,.or.other.temperature.control.device.for.2-ml.tubes Microcentrifuge Optional:.Ice-cold.100%.and.70%.ethanol.(see.pages.5.and.9.for.details) Optional:.RNA.purification.columns.(see.pages.5.and.9.for.details) 3

4 Performance Specifications and Quality Control A.Ribo-Zero.rRNA.Removal.Kit.(Human/Mouse/Rat).reaction.removes.>98%.of.28S,.18S,. and.5.8s.and.>95%.of.5s.rrnas.from.intact.human.reference.rna.as.assessed.by.qrt-pcr. before.and.after.a.ribo-zero.kit.reaction. 2. Introduction Advantages of the Ribo-Zero rrna Removal Kit (Human/Mouse/Rat) Compared to other rrna Removal Methods: 1). Removes.28S,.18S,.5.8S,.and.5S.rRNA.from.human,.mouse,.or.rat.total.RNA. 2). Highly.effective.rRNA.removal.with.either.high-quality.(intact).or.partially.degraded. RNA.samples. 3). Recovers.the.5.fragments.of.mRNA.from.compromised.RNA.samples. 4). Single-pass.process.yields.RNA.sufficiently.depleted.of.rRNA.for.RNA-Seq,.randomprimed.cDNA.synthesis,.and.other.applications. Summary of the Ribo-Zero rrna Removal Process: 1). Prepare.Ribo-Zero.Microspheres.. (15.minutes).. 2). Treat.1-5.μg.of.total.RNA.with.Ribo-Zero.rRNA.Removal.Solution.. (25.minutes).. 3). Incubate.the.reaction.with.the.Ribo-Zero.Microspheres.. (20.minutes).. 4). Purify.the.rRNA-depleted.samples.by.ethanol.precipitation. (90.minutes)..or.by.a.column-based.method.(user.provided). (15.minutes) 3. Preparation Maintaining an RNase-Free Environment: Ribonuclease.contamination.is.a.significant.concern.for.working.with.RNA..The.ubiquitous. RNase.A.is.a.highly.stable.and.active.ribonuclease.that.can.contaminate.any.laboratory. environment.and.is.present.on.human.skin..creating.an.rnase-free.work.environment. and.maintaining.rnase-free.solutions.is.critical.for.performing.successful.rrna.removal. reactions..therefore,.we.strongly.recommend.that.you: 1). Use.RNase-free.tubes.and.pipette.tips. 2). Always.wear.gloves.when.handling.samples.containing.RNA..Change.gloves.frequently. especially.after.touching.potential.sources.of.rnase.contamination.such.as.door.knobs,. pens,.pencils,.and.human.skin. 3). Always.wear.gloves.when.handling.kit.components..Do.not.pick.up.any.kit.component. with.an.ungloved.hand. 4). Keep.all.kit.components.tightly.sealed.when.not.in.use..Keep.all.tubes.containing.RNA. tightly.sealed.during.the.incubation.steps. 4 EPICENTRE Biotechnologies

5 Input Total RNA: A.kit.reaction.will.remove.the.28S,.18S,.5.8S,.and.5S.rRNA.from.to.1-5.μg.of.input.human,. mouse,.or.rat.total.rna..the.total.rna.preparation.should.be.free.of.salts.(e.g.,.mg 2+. or.guanidinium.salts),.and.organics.such.as.phenol.and.ethanol..we.recommend.that. the.sample.be.dissolved.in.rnase-free.water.or.te.buffer..use.table.1.to.determine.the. maximum.volume.in.which.the.total.rna.sample.can.be.dissolved.. Note: This table is replicated in the reaction protocol on page 7. Table 1. Volumes of Ribo-Zero Ribosomal RNA Removal Solution. 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 >2.5-5 μg 26 μl 10 μl Ribo-Zero rrna Removal Solution (Human/Mouse/Rat): The.volume.of.Ribo-Zero.rRNA.Removal.Solution.used.in.a.reaction.is.dependent.on.the. amount.of.input.total.rna.(table.1)..it is important to quantify the amount of total RNA in the sample as accurately as possible in order to use the appropriate amount of Ribo-Zero rrna Removal Solution in Step B. Ribo-Zero Microspheres: The.Ribo-Zero.Microspheres.must.be.washed.prior.to.use..It.is.critical.to.resuspend.the. Ribo-Zero.Microspheres.into.a.homogeneous.slurry.before.dispensing.them.into.the. 2-ml.Wash.Tube(s).(provided.in.the.kit)..The.best.way.to.resuspend.the.Microspheres.is. by.vigorous.vortex.mixing..the.microspheres.are.capable.of.withstanding.vigorous.vortex. mixing.and.remain.in.homogeneous.suspension.for.several.minutes.after.mixing..when. treating.multiple.total.rna.samples,.we.strongly.recommend.that.the.microspheres.be. prepared.separately.for.each.sample..do not.batch-wash.the.microspheres.for.multiple. samples. Purification of the rrna-depleted Sample: The.rRNA-depleted.sample.can.be.purified.by.ethanol.precipitation.or.column.methods. (see.page.9)..the.kit.includes.sodium.acetate.and.glycogen.for.ethanol.precipitation.. For.column-based.purification.methods,.we.recommend.using.the.RNA.Clean.&. Concentrator -5.columns.(Zymo.Research;.provided.by.the.user).or.other.column. appropriate.for.the.volume.and.amount.of.rna.in.the.rrna-depleted.sample. Yield of rrna-depleted RNA: The.yield.of.rRNA-depleted.RNA.from.a.Ribo-Zero.reaction.is.dependent.on.the.amount.of. input.total.rna,.the.rrna.content.of.the.sample,.and.the.method.used.to.purify.the.rrnadepleted.rna. 5

6 Familiarize Yourself with the Ribo-Zero rrna Removal Kit (Human/Mouse/Rat) and Procedure: Before.starting,.please.read.this.protocol.carefully.and.familiarize.yourself.with.each.kit. component.and.in.which.step.of.the.process.it.is.used...be.sure.to.wear.gloves.when.handling. the.kit.components. Control Reaction: We.recommend.that.those.who.are.not.experienced.with.the.Ribo-Zero.rRNA.Removal. Kit.(Human/.Mouse/Rat).perform.a.control.reaction.using,.for.example,.Human.Reference. RNA,.or.HeLa.or.NRK.total.RNA.(provided.by.the.user).prior.to.committing.a.precious. sample..to.correctly.evaluate.the.effectiveness.of.a.ribo-zero.reaction,.the.comparison. should.be.made.to.a.mock.control.ribo-zero.reaction.that.contains.the.starting.total.rna. minus.the.ribo-zero.rrna.removal.solution.and.is.otherwise.treated.in.the.same.manner. from.start.to.finish. 4. Ribo-Zero rrna Removal Kit (Human/Mouse/Rat) Procedure A. Prepare the Ribo-Zero Microspheres The.Ribo-Zero.Microspheres.must.be.washed.using.the.Ribo-Zero.Microsphere.Wash. Solution.and.then.resuspended.in.the.Ribo-Zero.Microsphere.Resuspension.Solution.before. use. Required.in.Step.A Component Name Tube Label Tube Color. Ribo-Zero.Microspheres. Microspheres. Colorless. Ribo-Zero.Microsphere.Wash.Solution. Microsphere.Wash.Solution. Green. Ribo-Zero.Microsphere.Resuspension.Solution. Resuspension.Solution. Red. RiboGuard.RNase.Inhibitor.(100.U/μl). RiboGuard.RNase.Inhibitor. Blue. Microsphere.Wash.Tube.(2.ml). Microsphere.Wash.Tube. Colorless 1.. Remove.the.Ribo-Zero.Core.Kit.from.4 C.storage.and.allow.the.tubes.to.warm.to.room. temperature..these.components.must.be.at.room.temperature.for.use.in.step.a3. Important! Allow the components of the Ribo-Zero Core Kit to equilibrate to room temperature for use in Step A Remove.the.Ribo-Zero.rRNA.Removal.Kit.(Human/Mouse/Rat).from. 70 C.to. 80 C. storage,.thaw.the.tubes,.and.place.them.on.ice. 3.. Vigorously.mix.the.room-temperature.Microspheres.for.20.seconds.by.vortexing.to. produce.a.homogeneous.suspension..the.microspheres.are.capable.of.withstanding. vigorous.vortex.mixing.and.remain.as.a.homogeneous.suspension.for.several.minutes. 4.. For.each.reaction,.pipette.65 μl of Microspheres.into.a.separate.2-ml.Microsphere.Wash. Tube..Aspirate.the.Microspheres.suspension.SLOWLY.to.avoid.air.bubbles.and.to.ensure. pipetting.the.full.required.volume..return.the.unused.microspheres.to.4 C. Important! Prepare the Microspheres for each RNA sample separately. DO NOT batchwash the Microspheres for multiple samples. 6 EPICENTRE Biotechnologies

7 5.. Centrifuge.the.dispensed.Microspheres.at.12,000.x.g.in.a.bench-top.microcentrifuge. for.3.minutes..remove.each.tube.from.the.microcentrifuge,.keeping.it.in.the.same. orientation.as.was.in.the.microcentrifuge,.and.carefully.pipette.off.and.discard.the. supernatant,.without.disturbing.the.microsphere.pellet.. Caution: The supernatant contains 0.1% sodium azide. Discard the supernatant according to local ordinances. 6.. Wash.the.Microspheres.by.adding.130 μl of Microsphere Wash Solution.to.each.tube.. Vigorously.vortex.(at.maximum.speed).the.tube(s).to.resuspend.the.Microspheres.. Centrifuge.the.tube(s).at.12,000.x.g.for.3.minutes.in.a.bench.top.microcentrifuge.. Remove.each.tube.from.the.microcentrifuge,.keeping.it.in.the.same.orientation.as.in. the.microcentrifuge,.and.carefully.pipette.off.and.discard.all.of.the.supernatant.without. disturbing.the.microsphere.pellet..7.. Add.65 μl of Microsphere Resuspension Solution.to.each.tube.and.resuspend.the. Microspheres.by.vigorous.vortex.mixing.at.maximum.speed.until.a.homogeneous suspension is produced. 8.. Add.1 μl of RiboGuard RNase Inhibitor.to.each.tube.of.resuspended.Microspheres.. Vortex.briefly.(10.seconds).and.store.the.tubes.at.room temperature.for.use.in.step.c. (page.8). B. Treat the Total RNA Sample with Ribo-Zero rrna Removal Solution Required.in.Step.B Component Name Tube Label Tube Color. Ribo-Zero.Reaction.Buffer. Ribo-Zero.Reaction.Buffer. Blue. Ribo-Zero.rRNA.Removal.Solution. rrna.removal.solution.(h/m/r). Blue....(Human/Mouse/Rat).. RNase-Free.Water. RNase-Free.Water. Colorless Additionally required for each reaction (provided by user): 0.2-ml.or.0.5-ml.microcentrifuge.tube.(RNase-free) Incubation temperatures performed in Step B:.68 C.and.room.temperature. 1.. 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.(see.table.below). 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 >2.5-5 μg 26 μl 10 μl 7

8 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 μg. Total.RNA.Sample.(see.Table.page.7). y. μl. Ribo-Zero.rRNA.Removal.Solution.(see.Table.page.7). 40. μl. Total.Volume 2.. Gently.mix.the.reaction(s).and.incubate at 68 C for 10 minutes..during.the.incubation. return.the.remaining.ribo-zero.rrna.removal.solution.and.ribo-zero.reaction.buffer. to.storage.at. 70 C.to. 80 C..Hint:.During.the.incubation,.familiarize.yourself.with.Step. C Remove.the.reaction.tube(s).and.incubate each at room temperature for 15 minutes. C. Microsphere Reaction and rrna Removal Required.in.Step.C:.50 C water bath or heating block.for.2.0-ml.tubes. 1.. Briefly.mix.by.vortexing.(at.medium.speed.for.about.20.seconds).the.washed,.roomtemperature.Microspheres.in.the.2.0-ml.Wash.Tube.from.Step.A8..If.necessary,. pulse-centrifuge.(5.seconds).to.collect.the.microsphere.suspension.in.the.bottom.of. the.tube,.then.resuspend.by.pipetting.the.slurry.several.times..it.is.important.to.have.a. homogeneous.slurry.before.adding.the.hybridized.rna.from.step.b Using.a.pipet,.add.the.hybridized.RNA.sample.from.Step.B3.to.the.resuspended. Microspheres.in.the.2.0-ml.Wash.Tube.and,.without changing the pipet tip, immediately.mix.the.contents.of.the.tube.by.rapidly.pipetting times..then,. immediately.mix.by.vortexing.(at.medium.speed).the.contents.of.the.tube.for.5.seconds. and.place.at.room temperature.before.proceeding.to.the.next.sample. Important! ALWAYS add the RNA sample to the resuspended Microspheres in the 2-ml Wash Tube and immediately and rapidly mix by pipetting the contents of the tube. NEVER add the Microspheres to the RNA sample. 3.. Incubate.the.tubes.at.room temperature for 10 minutes.with.vortex.mixing.(at.medium. speed).for.5.seconds.every.3.to.4.minutes. Important! DO NOT use a shaker platform as this does not provide sufficient mixing. 4.. At.the.end.of.the.10-minute.incubation.at.room.temperature,.mix.by.vortexing.(at. medium.speed).the.sample.for.5.seconds.and.then.place.at.50 C for 10 minutes.in.a. water.bath,.heating.block,.or.other.temperature-controlled.device. 5.. After.10.minutes.at.50 C,.immediately.transfer.the.RNA-microspheres.suspension.to.a. Microsphere.Removal.Unit.(filtration.unit;.provided.in.the.Ribo-Zero.Core.Kit.box).and. centrifuge.at.12,000.x.g.for.one.minute.at.room.temperature..save.the.eluate.that.is.in. the.collection.tube.and.discard.the.filter.unit.with.the.microspheres. Important! The eluate contains the rrna-depleted sample! 8 EPICENTRE Biotechnologies

9 D. Purify the rrna-depleted Sample The.rRNA-depleted.sample.can.be.purified.by.ethanol.precipitation.or.by.a.column.method.. Step.D,.Part.1.details.the.ethanol.precipitation.procedure.and.Step.D,.Part.2.provides. guidance.for.column.purification.using.a.rna.clean.&.concentrator-5.column.(zymo. Research;.Cat..Nos..R1015,.R1016). 1. Ethanol Precipitation of the rrna-depleted Sample. Component Name Tube Label Tube Color. RNase-Free.Water. RNase-Free.Water. Colorless. Sodium.Acetate.(3.M). Sodium.Acetate. Colorless. Glycogen.(10.mg/ml). Glycogen. Colorless Additionally Required for each reaction (provided by user): 1.5-ml.microcentrifuge.tube.(RNase-free) Ice-cold.70%.and.100%.Ethanol Incubation temperatures performed in Step D, Part 2:. 20 C Adjust.the.volume.of.each.sample.to.180.μl.using.RNase-Free.Water Add.18.μl.of.3.M.Sodium.Acetate.to.each.tube Add.2.μl.of.Glycogen.(10.mg/ml).to.each.tube.and.mix.by.gentle.vortexing Add.three.volumes.(600.μl).of.ice-cold.100%.ethanol.to.each.tube.and.mix.thoroughly. by.gentle.vortexing Place.the.tubes.at. 20 C.for.at.least.1.hour Centrifuge.the.tubes.at.>10,000.x.g.in.a.microcentrifuge.for.30.minutes..Carefully. remove.and.discard.the.supernatant Wash.the.pellet.with.ice-cold.70%.ethanol.and.centrifuge.at.>10,000.x.g.for.5.minutes.. Carefully.remove.and.discard.the.supernatant Repeat.Step.7.(above).one.more.time 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 Dissolve.the.pellet.in.the.desired.volume.of.RNase-Free.Water.or.buffer..The.rRNAdepleted.RNA.can.be.used.immediately.or.stored.at. 70 C.to. 80 C. 2. Column Purification of the rrna-depleted Sample. 1.. We.recommend.using.the.RNA.Clean.&.Concentrator-5.Column.(Zymo.Research).to. purify.the.rrna-depleted.rna.sample..follow.the.manufacturer s.procedure.best.suited. to.the.needs.of.the.downstream.application.of.the.rrna-depleted.rna.sample..for. example,.follow.the.zymo. General.Procedure,.total.RNA.(>17.nt).to.recover.all.the. rrna-depleted.rna.including.small.rna,.or.follow.the.zymo. Small.RNA.elimination,. total.rna.(>200.nt).procedure.to.recover.the.rrna-depleted.rna.if.greater.than.200.nts.. RNA.purification.columns.from.other.suppliers.may.also.be.used;.however,.performance. may.vary. 2.. The.eluted.RNA.can.be.used.immediately.or.stored.at. 70 C.to. 80 C. 9

10 5. Quantifying the Yield and Assessing the Quality of the rrna-depleted Sample The.yield.of.rRNA-depleted.RNA.from.a.Ribo-Zero.reaction.is.dependent.on.the.amount. of.input.total.rna,.the.rrna.content.of.the.sample,.and.the.method.used.to.purify.the. rrna-depleted.rna..the.yield.of.rrna-depleted.rna.can.be.determined.by,.for.example,. RiboGreen.RNA.Reagent.(Invitrogen).or.using.a.NanoDrop.Spectrophotometer.(Thermo. Scientific)..The.integrity.of.the.rRNA-depleted.sample.and.the.removal.of.the.28S.and.18S. rrnas.from.high.quality.(intact).total.rna..can.be.assessed.by.denaturing.agarose.gel. electrophoresis.or.by.using.an.agilent.2100.bioanalyzer.(figures.1a.and.b,.below). Please note that when using the Zymo column General Procedure, total RNA (>17 nt) for purifying the rrna-depleted RNA, small RNAs such as mirna and trna are not removed from the sample by the Ribo-Zero process. Therefore, the presence of a high proportion of small RNA in the rrna-depleted sample should not be interpreted as degradation of the RNA. A B Figure 1. Bioanalyzer profile of Ribo-Zero-treated human brain RNA and corresponding mrna- Seq library. A) Five micrograms of Human brain RNA was treated with the Ribo-Zero rrna Removal Kit (Human/Mouse/Rat) and the resulting rrna-depleted RNA was analyzed on a Bioanalyzer. B) Bioanlayzer profile of the mrna-seq library prepared from 50 ng of the Ribo-Zero treated RNA using EPICENTRE s ScriptSeq mrna-seq Library Preparation Kit (Illumina-compatible). 10 EPICENTRE Biotechnologies

11 6. Related Products Ribo-Zero rrna Removal Kit (Human/Mouse/Rat) Low Input RZH Reactions. For.50.ng-1.μg.of.input.total.RNA..Removes.>99%.of.the.rRNA.from.human,.mouse,.. and.rat.total.rna.preparations. Ribo-Zero rrna Removal Kit (Gram-Negative Bacteria) RZNB Reactions. Removes.>99%.of.the.rRNA.from.Gram-negative.bacteria.total.RNA.preparations. Ribo-Zero rrna Removal Kit (Gram-Positive Bacteria) RZPB Reactions. Removes.>99%.of.the.rRNA.from.Gram-positive.bacteria.total.RNA.preparations. ScriptSeq mrna-seq Library Preparation Kit (Illumina-compatible) SS Reactions SS Reactions ScriptSeq mrna-seq Library Preparation Kit (Roche Titanium-compatible) SST Reactions ExactSTART Eukaryotic mrna 5 - & 3 -RACE Kit ES Reactions ExactSTART Full-Length cdna Library Cloning Kit ES Reactions RiboMultiplier Sense-RNA Amplification Kit RM Reactions Ribo-Zero, RiboGuard, ScriptSeq, ExactSTART, and RiboMultiplier are trademarks of EPICENTRE, Madison, Wisconsin. RNA Clean & Concentrator is a trademark of Zymo Research, Orange, California. RiboGreen is a registered trademark of Molecular Research Center Inc., Cincinnati, Ohio. NanoDrop is a registered trademark of NanoDrop Technologies Inc., Wilmington, Delaware. Visit.our.technical.blog:.epicentral.blogspot.com 11

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