Laser Microdissection and RNA
Tips for
sample preparation, handling
and RNA-extraction
Dr. Ulrich Sauer, MicroImaging-Labs, München
Nothing But Light…
• Non-contact
• Against gravity
Contamination free
Catapulting
Laser Pressure
ZEISS
LD Plan-NEOFLUAR
40x / 0.6
421361-9970
Laser
Microdissection
ZEISS
LD Plan-NEOFLUAR
40x / 0.6
421361-9970
How does LCM work?
First Prerequisite: Good Recognition
Improved vision: Liquid Cover Glass (LCG)
LCG
open section
20x
AdhesiveCap opaque
Improved vision: Liquid Cover Glass (LCG)
LCG
LCG
open section
40x
AdhesiveCap opaque
PALM Consumables
Superior Performance for Fixed Material and Live Cell Applications
Fixed material
Live cells / tissue
LCM from kidney biopsy (FFPE, H&E)
What happens on a microtome?
Intrinsic RNase Activities (your worst enemy)
Intrinsic RNase activities can vary dramatically across different tissues
which points out the importance of strict RNase control.
Quantitative hierarchy of RNase activities in mouse tissues
(cited from Krosting J, Latham G, AMBION, Inc.)
Mouse tissue
Pancreas
Spleen
Lung
Liver
Thymus
Kidney
Heart
Brain
Fold increase relative to brain
181,000 x
10,600 x
5,300 x
64 x
16 x
8x
2x
1x
Standard RNA-protective Measures
Speed + temperature and dryness
Fixatives
RNAlater
DEPC- treatment (diethylpyrocarbonate)
Gloves, filter-tips, RNase- free materials
Detergents: SDS (= sodiumdodecylsulfate)
Autoclaving; baking at 180°C / 4 h
RNase-inhibitors (eg. RNasin)
RNase-away, RNase-Zap, etc.
Chaotropic solutions (GTC, GuHCl, Phenol)
Stainings
histochemical
(H&E, CresylViolet, methylgreen,...)
immunohistochemical
fluorescence (FITC, rhodamin...),
color reactions (HRP/AP, DAB, AEC...)
direct fluorescence label
GFP in transgenes, DAPI on DNA,
mitotracker, phalloidin-Alexa
RNA quality determination: Bioanalyzer
Agilent Application Note #5989-1165EN
RNA-Quality
28S
18S
Cryosection
Paraffin (FFPE)
RNA Integrity after Laser Microdissection
28s RNA
Agilent 2100 Bioanalyzer RNA quality check:
18s RNA
Starting with RNA from a large area:
mouse liver tissue (> 10.000 cells)
Down to a low number of cells
mouse liver tissue (~ 500 cells)
Recommended Staining Procedure for RNase-rich Tissue
For optimal protection of RNA take a pre-cooled MembraneSlide (best inside the cryostat
chamber at about -20°C) and touch the backside of the slide with your finger (wear gloves!)
to warm only the region for placing the section on the membrane.
Transfer the section from the blade to the slide by touching with the warmed area.
Dry in the cryostat for 2-3 min at about -20°C. (Longer drying may improve the adhesion of
sections if that is critical during the following staining steps.)
Subsequently incubate the section in ice-cold 70% ethanol for 2-3 min to reduce RNase
activity by dehydration.
Now incubate the section in ice-cold CresylViolet-staining solution for 30 sec to 2 min.
Remove excess stain from the slide on an absorbant surface, then dip few times into ice-cold
70% ethanol for short washing
Complete washing and dehydration by few dips into ice-cold 100% ethanol
Finally air-dry at room temperature for 1-2 minutes
After drying slides can be used immediately for LCM or may be stored at -80°C for some days
LCM of hair follicle for RNA (CresylViolet stain)
1
2
3
4
RNA-Extraction: frozen (Qiagen RNeasy Micro kit)
We recommend AdhesiveCaps (200 or 500) for collection of any RNA samples.
Our most safe staining procedure for frozen sections applies Cresyl Violet in 50%
ethanol and is performed in an ice bath.
Note: If excess OCT or another tissue freezing medium was used, an additional
washing step in ice-cold RNase-free water (1-2 min) has to be performed after
the first ethanol step.
Note: Slides should be frozen and thawed in an airtight container (e.g., two slides
back-to-back in a 50 ml Falcon-Tube). To avoid condensation of moisture on the
tissue do not open the container before slides are warmed up again to ambient
temperature. When sections are completely dry, RNases are inhibited and RNA
is remarkably stable for hours.
Note: The lysis in RLT should be performed „upside down“ for at least 30 minutes
for best efficiency and yield.
Effect of remaining OCT
Effect of staining and moisture
fresh
CV-stained
dry overnight
wet overnight
Effect of liquids at room temperature (like IHC)
dry (5 h)
PBS (2 h)
PBS (5 h)
dry (5 h)
2M NaCl (5 h)
Improved RNA preservation for
immunolabeling and laser microdissection
Amanda L. Brown and Doug W. Smith
RNA 2009 15: 2364-2374
originally published online October 22, 2009
Difficult RNA-material: pancreas tissue (frozen)
fresh (RIN 6.8)
„rest“ (RIN 3.1)
islets (RIN 8.1)
exocrine (4.6)
Selective LCM from RNAlater - treated tissue
Cryo section, human prostate duct, CresylViolet stain, 40x
• Selective elimination
of unwanted material
• Clear cut between
selected and unwanted
specimen
Schlomm T et al. Int J Oncol, 27, 3: 713-720 (2005)
Homogenous sample collection with LCM
Images by courtesy of Dr. T. Schlomm, UKE Hamburg
RNA analysis: Agilent Bioanalyzer results
Images by courtesy of Dr. T. Schlomm, UKE Hamburg
RNA-Extraction: formalin fixed (Qiagen RNeasy FFPE kit)
We recommend AdhesiveCaps (200) for collection of FFPE RNA-samples.
Note: For formalin fixed samples a Proteinase K digestion step is essential!
The time necessary for optimal Proteinase K digestion depends on many factors
like tissue type, fixation procedure or element size of lifted material.
An overnight digestion (12-18 hours) is a good starting point for optimization, but
shorter digestion times may be tested as well.
To our experience digestion should be performed for of at least 3 h at 56°C.
Note: The ProteinaseK digest should be performed „upside down“ for best efficiency.
Note: The use of random or gene-specific primers for RT is very important since
reverse transcription of formalin fixed RNA with only standard oligoT-primers will be
very inefficient and strongly 3`biased due to the numerous strand breaks and
modifications inflicted by the formalin fixation and paraffin embedding procedure.
RNA from FFPE (different Prot.K digestion times)
brain
gut
kidney
liver
Testing new RNA extraction procedures for FFPE
New Qiagen RNeasy FFPE
with DNase I on column digest
(replacing the gDNA eliminator column)
Qiagen Allprep FFPE (both DNA + RNA)
Testing new RNA extraction procedures for FFPE
„new“
„old“
rat kidney 500,000 µm²
Allprep workflow for DNA and RNA
FFPE-sample
Prot.K time!
Pellet
Supernatant
RNA
DNA
Allprep RNA: timecourse Prot.K digest
o.n.
RNeasyFFPE new, 4h
2
4
0.5
Allprep,1h
Allprep, 30min
Allprep, 2h
1
Allprep RNA + DNA
1h
1,5 h
45 min
1h
QiaAmp
o.N.
45 min
1,5 h
Laser Microdissection Overview
Bridging the gap from microscopy to molecular analysis
Preparation
DNA
Preparation
RNA
Proteins
Life cells
Greetings from MI-Labs in Munich!
[email protected]