Title: Isolating Total RNA?
1Isolating Total RNA? Scott Tighe 802-656-2557
HSRF 305
2RNA 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!
3Degradation 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
4Why 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
5General 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
6General 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
-
7Why an RNase Inhibitor?
Time-0
With RI
No RI
8RNases are almost everywhereResults from an
RNase test system
Surface treated RNase A followed by
Decontamination
Several RNase Inhibitors
9RNA Extraction Systems
10Silica 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
-
11RNA 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
12Trizol-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
13Hybrid 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
14Considerations 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
15Considerations 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
16Considerations 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
17Extracting RNA
18Extracting 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
19Extracting 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
20Extracting 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
21- 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
22Extracting 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
23Extracting 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)
24Quality Control of RNA
25Quality Control of RNA
- Consider running an RNase-free gel
- Look for gDNA
- Look for rRNA bands
- We use the E-gel and FlashGels
26- 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
27Good vs Degraded RNA
Thing are not always as they appear- These look
great on the nanodrop but. are they?
28Expected 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
29Problematic 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
30Nanodrop Spectra
EDTA GUAN-HCL RLT
Trizol GUAN-ISOThio Tween 20
31Nanodrop Spectra
Glycogen Amm. ace Sod. ace
PCI MES Salt Alginate
32Nanodrop Spectra
Residual Trizol ETOH
RNase Inhibitor Tris
33Round table discussion
34FACS and LCM
35FACS 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
36- 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
37LCM 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
38Scape 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
39Thank you for your attention!