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Isolating Total RNA?Scott Tighe

802-656-2557HSRF 305

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RNA StructureRNA StructureMajor TypesmRNA-transcriptionrRNA-

5s,5.8s,16s, 18s,23s,26s 28s

tRNA-Involved in PS

Other ncRNA-miRNA, siRNA snRNAsnoRNASmYscaRNAgRNARNase PRNase MRP

Different from DNA has 2’OH Group!

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Degradation of RNA-Two Major CatagoriesDegradation of RNA-Two Major Catagories

Yingfu Li, and Ronald R. Breaker J. Am. Chem. Soc., 1999, 121 (23), 5364-5372

Catalytic/ EnzymaticRNases

-Catalytic His 12 and His 119, produce a 2’-3’ cyclic phosphate intermediate similar to chemical catalysis

Ribozymes-RNase P

Chemical Catalysis byAcid, Base, Divalent Ion2’ oxygen attacks the adjacent phosphate 2’ OH transesterification

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Why Total RNA?Why Total RNA? Not all transcripts have poly A tail- ie mitochondrialNot all transcripts have poly A tail- ie mitochondrial

RNA assessment is more determinative RNA assessment is more determinative

rRNA subunit have decrete peaks when running a gel or BioanalyzerrRNA subunit have decrete peaks when running a gel or Bioanalyzer

Recovery of special RNA’s such as miRNA, NC RNA, nuclear RNA ectRecovery of special RNA’s such as miRNA, NC RNA, nuclear RNA ect

mRNA recovery kits also recover rRNA anywaymRNA recovery kits also recover rRNA anyway

Total RNA mRNA

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General RNA HandlingGeneral RNA HandlingReagents and Equipment-ConsiderationsReagents and Equipment-Considerations::

All reagents MUST be RNase-freeAll reagents MUST be RNase-free

Use gloves that are periodically treated with RNase ZapUse gloves that are periodically treated with RNase Zap

Perform all work in a hoodPerform all work in a hoodBiosafetyBiosafetyLaminar flow Laminar flow PCR hoodPCR hoodDO NOTDO NOT use a fume hood use a fume hood

Use aerosol resistant pipet tips ONLYUse aerosol resistant pipet tips ONLY

Prepare all surfaces and pipets by treating with RNAse ZapPrepare all surfaces and pipets by treating with RNAse Zap

All utensils [scissors, scalpels, tweezers] should be scrubbed clean, All utensils [scissors, scalpels, tweezers] should be scrubbed clean, sprayed with RNase Zap, soaked in ETOH and flame sterilized sprayed with RNase Zap, soaked in ETOH and flame sterilized before a surgerybefore a surgery

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Prepare daily aliquots of RNase-free water. I aliquot 10-20 tubes each week and discard half Prepare daily aliquots of RNase-free water. I aliquot 10-20 tubes each week and discard half way through the day.way through the day.

Diethylpyrocarbonate [DEPC]-treated water is NOT an inhibitor for RNases, but rather DEPC is Diethylpyrocarbonate [DEPC]-treated water is NOT an inhibitor for RNases, but rather DEPC is a chemical added to water to eliminate RNases. After autoclaving [or when purchased], no a chemical added to water to eliminate RNases. After autoclaving [or when purchased], no DEPC resides in the water.DEPC resides in the water.

When opening and closing tubes, be careful not to bump the inner rim of your tubes. When opening and closing tubes, be careful not to bump the inner rim of your tubes.

Use a RNase Inhibitor if allowable by Use a RNase Inhibitor if allowable by downstream reactionsdownstream reactions

RiboLockRiboLockSuperaseSuperaseothersothers

General RNA HandlingGeneral RNA Handling

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Why an RNase Inhibitor?Why an RNase Inhibitor?

RNase Test

Time-0

No RI With RI

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RNases are almost everywhereRNases are almost everywhere

Results from an RNase test systemResults from an RNase test system

Surface treated RNase A followed by Decontamination Several RNase Inhibitors

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RNA Extraction SystemsRNA Extraction Systems

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Most use 4M guanidine isothiocyanate [GCN] chaotropic salt Most use 4M guanidine isothiocyanate [GCN] chaotropic salt system that denatures RNases without the use of phenolsystem that denatures RNases without the use of phenol

RNA is precipitated with ethanol and bound to silica [Si-O-RNA is precipitated with ethanol and bound to silica [Si-O-H], washed, and eluted with water. H], washed, and eluted with water.

PROS PROS CONSCONSEasy to performEasy to perform Lipids will interfere and inhibitLipids will interfere and inhibitCompatible with Shedder column Compatible with Shedder column Will not isolate MiRNAWill not isolate MiRNA

No precipitation rxnNo precipitation rxn Will not isolate RNA <200bpWill not isolate RNA <200bpVery clean RNAVery clean RNA Lower yield than TrizolLower yield than TrizolCompatible with FastPrep Compatible with FastPrep Salts may be left behindSalts may be left behindDNase on the columnDNase on the column RLT Poor storage RLT Poor storage

integrity at -80integrity at -80Abrasives can end up in final Abrasives can end up in final

samplesampleheavy DNA contamination heavy DNA contamination

problemsproblems

Silica Column-basedSilica Column-based

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RNA Isolation and Purification Systems-RNA Isolation and Purification Systems-[Column-based][Column-based]

RNeasy Micro kit [74004] RNeasy Micro kit [74004] Small elution volumes 10-15ul for 10ugSmall elution volumes 10-15ul for 10ugGood for FACS samples, LCM, or limited cellGood for FACS samples, LCM, or limited cellOn-column DNase treatmentOn-column DNase treatment

RNeasy Mini Kit [74104]RNeasy Mini Kit [74104]Standard Elution volume of 30-50ul for 100ugStandard Elution volume of 30-50ul for 100ugGeneral use columnGeneral use columnOn column DNase treatmentOn column DNase treatment Lipid or Fiber kits tooLipid or Fiber kits too

RNeasy Midi and Maxi KitRNeasy Midi and Maxi KitLarge elution volumes 150-800ul for 1mg of RNALarge elution volumes 150-800ul for 1mg of RNA

USB Corp Prep-Easy Kit USB Corp Prep-Easy Kit

Invitrogen Pure Link microInvitrogen Pure Link micro

Ambion RNAqueousAmbion RNAqueous

Zymo Corp-Not recommendedZymo Corp-Not recommended

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Phenol-Guanidine reagentPhenol-Guanidine reagent

TriZolTriZol Tri-Reagent Tri-Reagent TriSureTriSure PurezolPurezol QiaZolQiaZol

Three typesThree types

Trizol – Standard-1:9 ratio must be maintained [10% sample]Trizol – Standard-1:9 ratio must be maintained [10% sample]Trizol LS -Concentrated-1:3 ratio must be maintained [33% sample]Trizol LS -Concentrated-1:3 ratio must be maintained [33% sample]Trizol BD-designed specifically for bloodTrizol BD-designed specifically for blood

Remove extracellular biomolecules with chloroform or bromochrolopropaneRemove extracellular biomolecules with chloroform or bromochrolopropane

Requires a precipitation reactionRequires a precipitation reaction

Use Axygen MCT175C ultra clear tubesUse Axygen MCT175C ultra clear tubesbetter pelletingbetter pelletingeasier to seeeasier to see

May add Pellet Paint, GlycoBlue to precipitation reaction for low [ ] of RNAMay add Pellet Paint, GlycoBlue to precipitation reaction for low [ ] of RNA

Trizol-based Reagents

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Hybrid SystemsHybrid Systems

Use both a Trizol (guanidine-phenol) reagent and silica Use both a Trizol (guanidine-phenol) reagent and silica columncolumn

Trizol Plus system [Invitrogen]Trizol Plus system [Invitrogen]

Qiagen RNeasy Lipid Tissue Mini KitQiagen RNeasy Lipid Tissue Mini Kit

Bio-Rad Aurum Total RNA Fatty and Fibrous Tissue KitBio-Rad Aurum Total RNA Fatty and Fibrous Tissue Kit

Home Made:Home Made: Perform the standard TrizolPerform the standard Trizol Transfer Aqueous phase to new tubeTransfer Aqueous phase to new tube Add 1.5x volume of 100% ETOHAdd 1.5x volume of 100% ETOH Apply to columnApply to column Follow standard column proceuduresFollow standard column proceudures

Sometimes leaves a Trizol residue on low recovery samplesSometimes leaves a Trizol residue on low recovery samples

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Considerations on which system to useConsiderations on which system to useTriZol ApplicationsTriZol Applications

Tissues high in lipid and other cellular biomolecules will require Tissues high in lipid and other cellular biomolecules will require Trizol because of potential interferences of the Si-OH Trizol because of potential interferences of the Si-OH

Trizol has a higher recovery of RNA then RNeasy columns but not Trizol has a higher recovery of RNA then RNeasy columns but not useful for very small amounts of RNA [+/-]useful for very small amounts of RNA [+/-]

Must be cleaned and DNase-treated using columnMust be cleaned and DNase-treated using column

Use of Bromochloropropane instead of chloroform for higher Use of Bromochloropropane instead of chloroform for higher purity. 260/280 ratios are often 1.6-1.8 with chloroform and 1.8-purity. 260/280 ratios are often 1.6-1.8 with chloroform and 1.8-2.0 for BCP.2.0 for BCP.

loss of the 28S subunit for solid tissueloss of the 28S subunit for solid tissue

Advantageous when grinding matrix is hard to remove- it spins Advantageous when grinding matrix is hard to remove- it spins outout

Can recover RNA and DNACan recover RNA and DNA

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Considerations on which system to use

Silica Column-based Applications [RNeasy]Silica Column-based Applications [RNeasy]

General use and rapidGeneral use and rapid

No precipitation reaction needNo precipitation reaction need

Optimized columns for very small concentrationsOptimized columns for very small concentrations

On-column one step DNase TreatmentOn-column one step DNase Treatment

No Phenols or organic solventsNo Phenols or organic solvents

Grinding matrix can end up in the sampleGrinding matrix can end up in the sample

Recoveries of approximately 60% of Trizol (we’ve seen)Recoveries of approximately 60% of Trizol (we’ve seen)

DNase-treatment will generally reduce yield by 30% or DNase-treatment will generally reduce yield by 30% or soso

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Considerations on which system to use

Silica Column-based Applications [RNeasy]…continuedSilica Column-based Applications [RNeasy]…continued

Use either centrifuge or vacuum manifold-Multiple loadingsUse either centrifuge or vacuum manifold-Multiple loadings

Heat elution water to 60C will increase yieldHeat elution water to 60C will increase yield

Do not spin with column openDo not spin with column open

260/280 ratios are often above 2.00260/280 ratios are often above 2.00

If using MinElute or MicroElute columns-be aware of the o-ring-it If using MinElute or MicroElute columns-be aware of the o-ring-it catches and retains liquid that can get into your final samplecatches and retains liquid that can get into your final sample

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Extracting RNAExtracting RNA

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Extracting RNA from TissueExtracting RNA from Tissue

RNA integrity is a function of tissue type and handling!RNA integrity is a function of tissue type and handling! Column or Trizol or ….Trizol follow by a column clean-upColumn or Trizol or ….Trizol follow by a column clean-up Know your tissue characteristics before startingKnow your tissue characteristics before starting

High RNase content tissuesHigh RNase content tissuesSpleenSpleenPancrease Pancrease IntestineIntestineThymusThymus

Connective tissues, collagen, protein, glycogen, lipids Connective tissues, collagen, protein, glycogen, lipids can interfere with silica columncan interfere with silica column

BrainBrainLiverLiverHeartHeartMuscleMuscleAdiposeAdipose

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Use fresh tissue when possibleUse fresh tissue when possible

Use a hoodUse a hood

Use only utensils that are disposable RNase-free or treated with RNase Use only utensils that are disposable RNase-free or treated with RNase Zap,ETOH, and flame sterilized!!!Zap,ETOH, and flame sterilized!!!

Take special precautions when working with challenging tissues such as Take special precautions when working with challenging tissues such as

glazing tissue with RNase inhibitorglazing tissue with RNase inhibitor

Place directly into extraction reagent and extract immediate [i.e. homogenize] Place directly into extraction reagent and extract immediate [i.e. homogenize] GCNGCN TriZolTriZol

DO NOT overload the extraction reagent DO NOT overload the extraction reagent Interferance of DNA, lipids or polysaccharide can inhibit silica reactionInterferance of DNA, lipids or polysaccharide can inhibit silica reaction

Extracting RNA from TissueExtracting RNA from Tissue

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Extracting RNA from Tissue- Extracting RNA from Tissue- Types of homogenizationTypes of homogenization Traditional mortor and pestle with LN2Traditional mortor and pestle with LN2

Liquid Nitrogen is Liquid Nitrogen is notnot always RNase-free always RNase-free Difficult to make sterile and RNase-freeDifficult to make sterile and RNase-free

Poly-tron with new tips or RNase-free bladesPoly-tron with new tips or RNase-free blades Not optimized for very small quantitiesNot optimized for very small quantities Sometimes difficult to make RNase-freeSometimes difficult to make RNase-free

Mini motorized pestleMini motorized pestle With or without abrasiveWith or without abrasive All RNase-free disposable All RNase-free disposable

Impactor [bio-pulverizer]Impactor [bio-pulverizer] French PressFrench Press Biomasher columnsBiomasher columns

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FastPrep System - Mini-bead beater FastPrep System - Mini-bead beater Automated, fast, RNase-freeAutomated, fast, RNase-free Uses screw cap 2ml tubes, 15ml and 50ml and Uses screw cap 2ml tubes, 15ml and 50ml and optional abrasive for homogenizing tough tissueoptional abrasive for homogenizing tough tissue Excellent for bacterial extractionsExcellent for bacterial extractions

In Room HSRF 305-Sign upIn Room HSRF 305-Sign up

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Extracting RNA from adherent cellsExtracting RNA from adherent cells

Directly from plate or dishDirectly from plate or dish

Similar for both Trizol and RNeasy systemSimilar for both Trizol and RNeasy system

Do not trypsinizeDo not trypsinize

Remove growth media and washing with PBS (with or w/o RI)Remove growth media and washing with PBS (with or w/o RI)

Add enough extraction reagent fro the number of cellsAdd enough extraction reagent fro the number of cells

Vortex plate directlyVortex plate directly

Follow standard operating procedureFollow standard operating procedure

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Extracting RNA from other SourcesExtracting RNA from other SourcesSuspended mammalian cells Suspended mammalian cells

Centrifuge to a pelletCentrifuge to a pellet

Wash with PBS(RI) to remove serum and mediaWash with PBS(RI) to remove serum and media

Add extraction reagent and vortex Add extraction reagent and vortex

Follow SOPFollow SOP

Viability is key to recovering intact RNA- Trypan or EosinViability is key to recovering intact RNA- Trypan or Eosin

Bacteria and yeast RNA’s are best recovered during early log Bacteria and yeast RNA’s are best recovered during early log phase!phase!

Heating of Trizol or RLT buffer to 50CHeating of Trizol or RLT buffer to 50C May require FastPrep with abrasiveMay require FastPrep with abrasive Cell wall digestion (Lyticase, Prok, Lysozyme)Cell wall digestion (Lyticase, Prok, Lysozyme)

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Quality Control of RNAQuality Control of RNA

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Consider running an RNase-free gelConsider running an RNase-free gel Look for gDNA Look for gDNA Look for rRNA bandsLook for rRNA bands

We use the E-gel and FlashGels We use the E-gel and FlashGels

Quality Control of RNAQuality Control of RNA

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Measure your RNA on the Nanodrop and BioanalyzerMeasure your RNA on the Nanodrop and Bioanalyzer

Can not distinguish DNA from RNACan not distinguish DNA from RNA Can not distinguish degraded RNA from “good” RNACan not distinguish degraded RNA from “good” RNA

Good RNA

RNA prep with mostly gDNA from Neutrophils

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Good vs Degraded RNAGood vs Degraded RNA

Thing are not always as they appear- These look great on the nanodrop… but…. are they?

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Expected yield is very important!!!Expected yield is very important!!!

Just getting “enough” RNA is not always okJust getting “enough” RNA is not always ok

Actively growing mammalian cells contain 1-10 Actively growing mammalian cells contain 1-10 pg/cellpg/cell

Calculate your expected RNA recoveryCalculate your expected RNA recovery

If you are way below your expected yield than….If you are way below your expected yield than…. Selectively recovered RNA from weak or apoptotic Selectively recovered RNA from weak or apoptotic

cellscells Selectively recovered RNA from G0 or G2MSelectively recovered RNA from G0 or G2M Will significantly impact gene expression dataWill significantly impact gene expression data

Expected YieldExpected Yield

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Problematic Nanodrop TracesProblematic Nanodrop Traces

Sample is NOT 183 ng/ul Actually 38ng/ul as per Qubit Spectrofluoromater

272 nm peak is skewing data

260 272

How will this affect downstream processes such as RT-qPCR if one assumes equal RNA input to a cDNA reaction?

TRZ

RNA

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Nanodrop SpectraNanodrop Spectra

EDTA

GUAN-HCL

RLT

Trizol

GUAN-ISOThio

Tween 20

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Glycogen

Amm. ace

Sod. ace

PCI

MES Salt

Alginate

Nanodrop SpectraNanodrop Spectra

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Residual Trizol

ETOH

RNase Inhibitor

Tris

Nanodrop SpectraNanodrop Spectra

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Round table discussionRound table discussion

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FACS and LCMFACS and LCM

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FACS sorted cells and RNAFACS sorted cells and RNA

1] FACS must be RNase-free by bleach and other treatments1] FACS must be RNase-free by bleach and other treatments

2] Bacterial contamination in sheath tanks and dip tubes can 2] Bacterial contamination in sheath tanks and dip tubes can cause RNasescause RNases

3] Hold back cells –check viability- extract RNA as a pre-sort 3] Hold back cells –check viability- extract RNA as a pre-sort controlcontrol

4] Add Superase or RiboLock to sort tube and pre-sort tube 4] Add Superase or RiboLock to sort tube and pre-sort tube containing cellscontaining cells

5] Use RNase-free tubes to sort5] Use RNase-free tubes to sort

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6] Sort into an 6] Sort into an exact volumeexact volume of ice cold extraction reagent of ice cold extraction reagent Trizol LS Trizol LS RLT buffer [RNeasy Micro]RLT buffer [RNeasy Micro] Media or buffer with or without SuperaseMedia or buffer with or without Superase

7] After sorting measure the total volume and add the 7] After sorting measure the total volume and add the necessary volume of reagent to obtain the correct ratio of necessary volume of reagent to obtain the correct ratio of extraction reagent to sort liquid extraction reagent to sort liquid

8] Consider acceptable sort volumes8] Consider acceptable sort volumes

9] Extract immediately-do not store cells in extraction buffer 9] Extract immediately-do not store cells in extraction buffer at -80at -80

10] DNase-treatment causes RNA losses using RNeasy10] DNase-treatment causes RNA losses using RNeasy

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LCM and RNALCM and RNA

Test tissue section before starting by aseptically removing and extracting a Test tissue section before starting by aseptically removing and extracting a small section. DO NOT LET FROZEN TISSUE THAW!small section. DO NOT LET FROZEN TISSUE THAW!

FFPE tissues often yield no usable RNA and testing its condition before the FFPE tissues often yield no usable RNA and testing its condition before the start is a good ideastart is a good idea

Know what level of degradation your downstream application can tolerateKnow what level of degradation your downstream application can tolerate

Fresh frozen tissues perform wellFresh frozen tissues perform well

Use RNase Zap-ETOH treated microtome with new blade during sectioningUse RNase Zap-ETOH treated microtome with new blade during sectioning

Use RNase –free slides, tweezers, materialsUse RNase –free slides, tweezers, materials

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Scape a small section from the prepared slide and extract as a controlScape a small section from the prepared slide and extract as a control

Prepare all staining and dehydration reagents from RNase-free reagents and Prepare all staining and dehydration reagents from RNase-free reagents and DEPC waterDEPC water

Collect one LCM cap from large area of cells as a control-non-specific cellsCollect one LCM cap from large area of cells as a control-non-specific cells

Collect target cells and transfer cap to tube containing extraction agent-vortexCollect target cells and transfer cap to tube containing extraction agent-vortex

Extract immediatelyExtract immediately

We have had good luck with RNeasy micro and Pico Pure kitsWe have had good luck with RNeasy micro and Pico Pure kits

Omit DNase step to increase recovery when allowableOmit DNase step to increase recovery when allowable

Extract several caps into one extract buffer or several extract buffers and Extract several caps into one extract buffer or several extract buffers and combine to increase yieldcombine to increase yield

LCMLCM

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Thank you for your attention!Thank you for your attention!