Isolating Total RNA?

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Isolating Total RNA? Scott Tighe 802-656-2557
HSRF 305
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RNA Structure
Major Types mRNA-transcription rRNA- 5s,5.8s,16s,
18s,23s,26s 28s tRNA-Involved in PS Other
ncRNA- miRNA, siRNA snRNA snoRNA SmY scaRN
A gRNA RNase P RNase MRP
Different from DNA has 2OH Group!
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Degradation of RNA-Two Major Catagories
Catalytic/ Enzymatic RNases -Catalytic His
12 and His 119, produce a 2-3 cyclic
phosphate intermediate similar to chemical
catalysis Ribozymes-RNase P Chemical
Catalysis by Acid, Base, Divalent Ion 2 oxygen
attacks the adjacent phosphate 2 OH
transesterification
Yingfu Li, and Ronald R. Breaker J. Am. Chem.
Soc., 1999, 121 (23), 5364-5372
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Why Total RNA?

• Not all transcripts have poly A tail- ie
  mitochondrial
• RNA assessment is more determinative
• rRNA subunit have decrete peaks when running a
  gel or Bioanalyzer
• Recovery of special RNAs such as miRNA, NC RNA,
  nuclear RNA ect
• mRNA recovery kits also recover rRNA anyway

Total RNA mRNA
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General RNA Handling

• Reagents and Equipment-Considerations
• All reagents MUST be RNase-free
• Use gloves that are periodically treated with
  RNase Zap
• Perform all work in a hood
• Biosafety
• Laminar flow
• PCR hood
• DO NOT use a fume hood
• Use aerosol resistant pipet tips ONLY
• Prepare all surfaces and pipets by treating with
  RNAse Zap
• All utensils scissors, scalpels, tweezers
  should be scrubbed clean, sprayed with RNase Zap,
  soaked in ETOH and flame sterilized before a
  surgery

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General RNA Handling

• Prepare daily aliquots of RNase-free water. I
  aliquot 10-20 tubes each week and discard half
  way through the day.
• 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 DEPC
  resides in the water.
• When opening and closing tubes, be careful not to
  bump the inner rim of your tubes.
• Use a RNase Inhibitor if allowable by
• downstream reactions
• RiboLock
• Superase
• others

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Why an RNase Inhibitor?
Time-0
With RI
No RI
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RNases are almost everywhereResults from an
RNase test system
Surface treated RNase A followed by
Decontamination
Several RNase Inhibitors
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RNA Extraction Systems
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Silica Column-based

• Most use 4M guanidine isothiocyanate GCN
  chaotropic salt system that denatures RNases
  without the use of phenol
• RNA is precipitated with ethanol and bound to
  silica Si-O-H, washed, and eluted with water.
• PROS CONS
• Easy to perform Lipids will interfere and
  inhibit
• Compatible with Shedder column Will not
  isolate MiRNA
• No precipitation rxn Will not isolate RNA
  lt200bp
• Very clean RNA Lower yield than Trizol
• Compatible with FastPrep Salts may be left
  behind
• DNase on the column RLT Poor storage
  integrity at -80
• Abrasives can end up in final sample
• heavy DNA contamination problems

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RNA Isolation and Purification Systems-Column-bas
ed

• RNeasy Micro kit 74004
• Small elution volumes 10-15ul for 10ug
• Good for FACS samples, LCM, or limited cell
• On-column DNase treatment
• RNeasy Mini Kit 74104
• Standard Elution volume of 30-50ul for 100ug
• General use column
• On column DNase treatment
• Lipid or Fiber kits too
• RNeasy Midi and Maxi Kit
• Large elution volumes 150-800ul for 1mg of RNA
• USB Corp Prep-Easy Kit
• Invitrogen Pure Link micro

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Trizol-based Reagents

• Phenol-Guanidine reagent
• TriZol Tri-Reagent
• TriSure Purezol QiaZol
• Three types
• Trizol Standard-19 ratio must be maintained
  10 sample
• Trizol LS -Concentrated-13 ratio must be
  maintained 33 sample
• Trizol BD-designed specifically for blood
• Remove extracellular biomolecules with chloroform
  or bromochrolopropane
• Requires a precipitation reaction
• Use Axygen MCT175C ultra clear tubes
• better pelleting
• easier to see

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Hybrid Systems

• Use both a Trizol (guanidine-phenol) reagent and
  silica column
• Trizol Plus system Invitrogen
• Qiagen RNeasy Lipid Tissue Mini Kit
• Bio-Rad Aurum Total RNA Fatty and Fibrous Tissue
  Kit
• Home Made
• Perform the standard Trizol
• Transfer Aqueous phase to new tube
• Add 1.5x volume of 100 ETOH
• Apply to column
• Follow standard column proceudures
• Sometimes leaves a Trizol residue on low recovery
  samples

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Considerations on which system to use

• TriZol Applications
• Tissues high in lipid and other cellular
  biomolecules will require Trizol because of
  potential interferences of the Si-OH
• Trizol has a higher recovery of RNA then RNeasy
  columns but not useful for very small amounts of
  RNA /-
• Must be cleaned and DNase-treated using column
• Use of Bromochloropropane instead of chloroform
  for higher purity. 260/280 ratios are often
  1.6-1.8 with chloroform and 1.8-2.0 for BCP.
• loss of the 28S subunit for solid tissue
• Advantageous when grinding matrix is hard to
  remove- it spins out
• Can recover RNA and DNA

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Considerations on which system to use
Silica Column-based Applications
RNeasy General use and rapid No
precipitation reaction need Optimized columns
for very small concentrations On-column one
step DNase Treatment No Phenols or organic
solvents Grinding matrix can end up in the
sample Recoveries of approximately 60 of
Trizol (weve seen) DNase-treatment will
generally reduce yield by 30 or so
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Considerations on which system to use
Silica Column-based Applications
RNeasycontinued Use either centrifuge or
vacuum manifold-Multiple loadings Heat elution
water to 60C will increase yield Do not spin
with column open 260/280 ratios are often
above 2.00 If using MinElute or MicroElute
columns-be aware of the o-ring-it catches and
retains liquid that can get into your final
sample
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Extracting RNA
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Extracting RNA from Tissue

• RNA integrity is a function of tissue type and
  handling!
• Column or Trizol or .Trizol follow by a column
  clean-up
• Know your tissue characteristics before starting
• High RNase content tissues
• Spleen
• Pancrease
• Intestine
• Thymus
• Connective tissues, collagen, protein, glycogen,
  lipids can interfere with silica column
• Brain
• Liver
• Heart
• Muscle
• Adipose

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Extracting RNA from Tissue

• Use fresh tissue when possible
• Use a hood
• Use only utensils that are disposable RNase-free
  or treated with RNase Zap,ETOH, and flame
  sterilized!!!
• Take special precautions when working with
  challenging tissues such as
• glazing tissue with RNase inhibitor
• Place directly into extraction reagent and
  extract immediate i.e. homogenize
• GCN
• TriZol
• DO NOT overload the extraction reagent
• Interferance of DNA, lipids or polysaccharide
  can inhibit silica reaction

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Extracting RNA from Tissue- Types of
homogenization

• Traditional mortor and pestle with LN2
• Liquid Nitrogen is not always RNase-free
• Difficult to make sterile and RNase-free
• Poly-tron with new tips or RNase-free blades
• Not optimized for very small quantities
• Sometimes difficult to make RNase-free
• Mini motorized pestle
• With or without abrasive
• All RNase-free disposable
• Impactor bio-pulverizer
• French Press
• Biomasher columns

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• FastPrep System - Mini-bead beater
• Automated, fast, RNase-free
• Uses screw cap 2ml tubes, 15ml and 50ml and
• optional abrasive for homogenizing tough tissue
• Excellent for bacterial extractions
• In Room HSRF 305-Sign up

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Extracting RNA from adherent cells

• Directly from plate or dish
• Similar for both Trizol and RNeasy system
• Do not trypsinize
• Remove growth media and washing with PBS (with or
  w/o RI)
• Add enough extraction reagent fro the number of
  cells
• Vortex plate directly
• Follow standard operating procedure

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Extracting RNA from other Sources

• Suspended mammalian cells
• Centrifuge to a pellet
• Wash with PBS(RI) to remove serum and media
• Add extraction reagent and vortex
• Follow SOP
• Viability is key to recovering intact RNA-
  Trypan or Eosin
• Bacteria and yeast RNAs are best recovered
  during early log phase!
• Heating of Trizol or RLT buffer to 50C
• May require FastPrep with abrasive
• Cell wall digestion (Lyticase, Prok, Lysozyme)

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Quality Control of RNA
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Quality Control of RNA

• Consider running an RNase-free gel
• Look for gDNA
• Look for rRNA bands
• We use the E-gel and FlashGels

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• Measure your RNA on the Nanodrop and Bioanalyzer
• Can not distinguish DNA from RNA
• Can not distinguish degraded RNA from good RNA

Good RNA
RNA prep with mostly gDNA from Neutrophils
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Good 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

• Expected yield is very important!!!
• Just getting enough RNA is not always ok
• Actively growing mammalian cells contain 1-10
  pg/cell
• Calculate your expected RNA recovery
• If you are way below your expected yield than.
• Selectively recovered RNA from weak or apoptotic
  cells
• Selectively recovered RNA from G0 or G2M
• Will significantly impact gene expression data

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Problematic Nanodrop Traces
Sample is NOT 183 ng/ul Actually 38ng/ul as per
Qubit Spectrofluoromater 272 nm peak is skewing
data
260 272
RNA
How will this affect downstream processes such as
RT-qPCR if one assumes equal RNA input to a cDNA
reaction?
TRZ
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Nanodrop Spectra
EDTA GUAN-HCL RLT
Trizol GUAN-ISOThio Tween 20
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Nanodrop Spectra
Glycogen Amm. ace Sod. ace
PCI MES Salt Alginate
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Nanodrop Spectra
Residual Trizol ETOH
RNase Inhibitor Tris
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Round table discussion
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FACS and LCM
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FACS sorted cells and RNA

• 1 FACS must be RNase-free by bleach and other
  treatments
• 2 Bacterial contamination in sheath tanks and
  dip tubes can cause RNases
• 3 Hold back cells check viability- extract RNA
  as a pre-sort control
• 4 Add Superase or RiboLock to sort tube and
  pre-sort tube containing cells
• 5 Use RNase-free tubes to sort

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• 6 Sort into an exact volume of ice cold
  extraction reagent
• Trizol LS
• RLT buffer RNeasy Micro
• Media or buffer with or without Superase
• 7 After sorting measure the total volume and add
  the necessary volume of reagent to obtain the
  correct ratio of extraction reagent to sort
  liquid
• 8 Consider acceptable sort volumes
• 9 Extract immediately-do not store cells in
  extraction buffer at -80
• 10 DNase-treatment causes RNA losses using
  RNeasy

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LCM and RNA

• Test tissue section before starting by
  aseptically removing and extracting a small
  section. DO NOT LET FROZEN TISSUE THAW!
• FFPE tissues often yield no usable RNA and
  testing its condition before the start is a good
  idea
• Know what level of degradation your downstream
  application can tolerate
• Fresh frozen tissues perform well
• Use RNase Zap-ETOH treated microtome with new
  blade during sectioning
• Use RNase free slides, tweezers, materials

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Scape a small section from the prepared slide and
extract as a controlPrepare all staining and
dehydration reagents from RNase-free reagents and
DEPC waterCollect one LCM cap from large area
of cells as a control-non-specific cellsCollect
target cells and transfer cap to tube containing
extraction agent-vortexExtract immediatelyWe
have had good luck with RNeasy micro and Pico
Pure kitsOmit DNase step to increase recovery
when allowableExtract several caps into one
extract buffer or several extract buffers and
combine to increase yield
LCM
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Thank you for your attention!