Laser capture microdissection=molecular analysis of ... Genomic Sampling... · microtomy and cryomicrotomy. • RNAse-free conditions: - Wipe everything (including brushes, inside

Disclaimers

Reference to any specific commercial products,

processes, manufacturers or company does not

constitute endorsement or recommendation by the

U.S. Government (the National Cancer Institute and

the National Institute of Environmental Health

Sciences) or SAIC Frederick. NIH/NCI/NIEHS and

SAIC Frederick are not responsible for any equipment,

software or related technical problems demonstrated

in this presentation. This presentation is intended for

educational purposes only.

Funded by NCI Contract HHSN261200800001E

Workshop Outline

• LCM sample is a genomic sample.

• Clinical vs experimental LCM sample.

•Genomic sample collection and stabilization.

•LCM sample preparation: microtomy, cryotomy, staining.

•Start-up QC of LCM sample.

•Storage of LCM sample.

•HANG ON- IT’S GOING TO BE AN INTENSE 45 MINUTES!

Laser Capture Microdissection=molecular

analysis of specific cells

Tissue

Tissue section

Specific cells Cell lysate

RNA

DNA

Protein

Molecular analysis of LCM sample

Slide thanks to Molecular Devices (modified)

Ciphergen

Gene Expression

MicroarrayMutation Analysis

Real-Time PCR

Gene

Expression

Analysis

Proteomics – Protein

Chip/Mass Spec

Proteomic

2D Gels

RNA, DNA

Protein

retrieved from

LCM sample

LCM sample=Genomic Sample

Intact RNA, DNA, Proteins are crucial for valid molecular analysis.

Take care of their integrity during all the steps.

Collection

•necropsy

•biopsy

Stabilization

•fixation

•freezing

Preparation for analysis

•OCT block (fresh or fixed tissue)

•Paraffin block (fixed tissue)

processing and embedding

•LCM slide preparation and

dissection

•Extraction (RNA,DNA,proteins)

Start Fresh and Sterile

Enzymes, naturally found in tissue, are reactivated unless desiccated and work during LCM to destroy your proteintargets (including DNA and RNA). This is a BAD thing if you are looking for DNA, RNA or other protein information!

Time, temperature and water are three factors to control when handling samples for LCM and analyzing for molecular data.

Fresher and Faster!!!!!!!

Clinical and Experimental LCM

Sample

Clinical sample

(human biopsy)

Preanalytical variability is

difficult to control.

Main focus on sample preparation for retrieval, and analysis ofcompromised nucleic acids and proteins in the sample

Experimental sample

(model animals)

Standard conditions and methods to minimize preanalytical variability in gene expression

Main focus on sample

collection and preparation

to preserve integrity of

intact nucleic acids and proteins in the sample

Preservation of RNA Integrity in Target

Organs During Necropsy.

• RNAse-free set-up.

• Separate instruments for

separate tissues.

• RNAse-free fixative.

• Priority of tissue removal

based on RNA stability.

• Stabilization of dissected

tissues (freezing, fixation or

incubation in RNA-later)

within 6 minutes after

euthanasia.

Targets are different; tailor necropsy

protocol to molecular endpoint

Stability of RNA in organs of

10 week old C57BL/6J Mice

Tissue RNA stability

Brain (whole)

Liver

stable

Lung

Prostate

unstable

Parotid

Pancreas

very unstable

extremely

unstable

15’ after euthanasia

Mouse liver

Mouse prostate

Six Minute Procedure for Multiple Tissue Harvest

(female)

Study objective: collection of 15 tissues (xiphoid process, spleen, kidney's cortex and

medulla layers, reproductive fat, ovaries, bladder, lung, thymus, heart and heart atria,

trachea, esophagus, pituitary and thyroid/parathyroid complex) for RNA retrieval.

Dissector #1

Euthanizes animals

Cuts tissues in RNAlater®

following its dissection

Assists the needs of the

dissector #2.

Dissector #2

Necropsy:

•lung, thymus, heart, trachea,

esophagus with a lower jaw

―in block‖

•head, spleen, kidney, reproductive fat,

ovaries and bladder

Distribution: tissues for sub-dissection.

Dissector #3

Separates the lower jaw and ,

passes it to dissector #4

Necropsy:

esophagus, heart, atria, trachea

and lung

Dissector #5

Removes pituitary out

of the head

Sub-dissects kidney

Dissector #4

Separates;

thyroid/parathyroid

complex off the

trachea.

Dissector #6

Supervises the dissections

through out the entire necropsy

session (time tracking, necropsy

documentation, etc.)

Cleans ovaries, xiphoid process,

and bladder from contaminating

tissue under dissecting

microscope.

Fulfilling the Requirements: Frozen vs. Fixed

SampleRequirements Fixed sample Frozen sample

Morphology excellent Depends on a tissue

type (from very good

to poor)

Suitability for IHC Depends on fixation type and conditions suitable

DNA Degradation level depends on fixation,

processing and storage

Could be chemically altered

Intact, suitable for all

downstream

applications

RNA Often significantly degraded.

Degradation level depends on fixation,

processing and storage and tissue type.


downstream

applications

Proteins Cross-linked, need specific retrieval

Suitable for some methods of protein

analysis.


methods of protein

analysis

In-situ hybridization Depends on a fixative and RNA integrity

in a section. Good to poor rate of

success.

High rate of success

Laser Capture

Microdissection

DNA is suitable for some applications

RNA is significantly degraded, suitable

for some applications. Low yield.

Good to poor rate of success.


applications

Slightly degraded,

suitable for all

applications

Sterile Blades

•New bottle of OCT freezing media.

•Sterilized forceps, blades, molds (RNA).

•Metal plate on dry ice for even freezing.

•Slides with frozen sections kept in dry ice while cutting.

•Store in -80ºC ultra-freezer and use within one week.

NIH/NIEHS website: www.niehs.nih.gov/research/atniehs/labs/lep/special/laser/index.cfm

Fresh frozen

sample is optimal.

RNAlater® as RNA Stabilizer.

• RNAlater® is a saturated salt solution that rapidly permeates fresh tissue and protects RNA from degradation.

Fresh frozen human kidney

biopsy after LCMRNAlater preserved biopsy

after LCM

Preservation of RNA Integrity in

Target Organs During Fixation.

• Optimal trim <= 3mm

• Freshly prepared fixatives

• Low temperature during fixation if possible

• RNAse-free reagents and conditions

• Short fixation (4-6 hr –4%PFA, 1.5-2 hr-modified Carnoy’s, 12-24 hr- 10% NBF) on a shaker.

Automated Processor Short Cycle

for Genomic Sample

4% PFA Modified Carnoy’s100% ETOH 20’, RT,on a

shaker, move into a processor

100% ETOH 20’, RT

100% ETOH 20’, RT

Xylene 20’, RT

Xylene 20’, RT

Paraplast 20’, 60ºC



85% ETOH 10’, RT

95% ETOH 15’, RT

95% ETOH 15’, RT

100% ETOH 10’, RT

100% ETOH 20’, RT

100% ETOH 20’, RT

Xylene 20’, RT

Xylene 20’, RT




RNA Quality from Paraffin Blocks

1. Archival Sample 10% NBF

2. Carnoy’s

3. 4% Paraformaldehyde

RIN=3.1 RIN=8.5

RIN=8.8

1 2

3

Preservation of RNA Integrity during

microtomy and cryomicrotomy.

• RNAse-free conditions:

- Wipe everything (including brushes, inside surface of the chamber and outer surfaces of the cryostat, and microtome surfaces) with 100% alcohol.

- Install a new disposable blade for each sample.

- RNAse-free water bath, individual container for block soaking.

• Facing the block, discard 5 first sections.

• Use anti-roll plate for cryo sections and automated microtome for paraffin sections.

• Move cryo sections on dry ice immediately after cutting (don’t dry).

• Dry paraffin sections at +56 C for 2 hours, cool at room temperature, and. Transfer to -80 C storage until use.

Mounting of OCT Section on PET

Slide

1. Flat Section Beneath the Anti-Roll Plate 2. Anti-roll plate removed, section should be

detached from the knife and attached to the

metal plate by gentle pressure.

3. Cool a UV- treated PET slide and immediately pick up the section by the

membrane window, touching the membrane with your finger.

NIEHS Whole Lysis Procedure

1. Pipette 50 µl of lysis

buffer on to sample.

2. Wait 30 seconds.

3. Pipette buffer back into tip.

4. Dispense into a 200-500 µl

microtube

Is RNA good for the desired downstream?

Sample Frozen (OCT) block

LCM cells

Mouse ileum 10 8.5 Mouse ileum 10 8.3 Mouse ileum 9.6 8.7 Mouse ileum 9.6 7.9 Mouse ileum 9.6 7.8 Mouse ileum 9.6 8.5 Mouse ileum 9.5 9.1 Mouse ileum 9.5 8.3 Mouse ileum 9.5 8.0 Mouse ileum 8.5 7.6 Mouse stomach 9.5 6.7 Mouse stomach 9.4 6.7 Human colorectal biopsy

9.2 7.0

Human skin biopsy

9.5 6.3

Human skin biopsy

9.4 6.7

Human skin 7.5 5.3

RNA integrity (RIN)

before and after LCM.

any application

Q-PCR

nanostring

RIN ≥ 8

RIN ≥ 7 microarray

RT-PCR

RIN ≤ 5

Critical Points of the LCM Staining

Protocol

• Exact timing of staining steps.

• Water- push it out of the protocol!

• Stain not more then 4-6 slides at a time.

• Slide storage in xylene (up to 2 hours) and in a

desiccator (up to 1 hour).

Evaluation of an LCM staining protocol

1. 100% ETOH (100 ml): acetic acid (3 ml)—20C , 30 sec

2. methyl green (vector)- 1000 ul + 2 ul RNase inhibitor- 40‖, drain slide on the kimwipe.

3. methyl green (vector)- 1000 ul + 2 ul RNase inhibitor- 40‖, drain slide on the kimwipe.

4. Apply 100 ul of cresyl violet/ eosin mix (300 cresyl+100 eosin+ 400 nuclease free water. Spin down before use, pipette from surface, don’t pick the last 50 ul from the tube) - 10‖.

5. 100% ETOH to rinse stain off (needs to be changed after 3 slides), flip gently once.

6. 100% ETOH-RT, 30 sec. 7. 100% ETOH-RT, 30 seconds 8. Xylene-RT, flip once, 2 min, 9. Xylene-RT, 3 min, 10. Airdry in the hood- 5 min

Scrap from the stained slide

RIN=7.7

RNA extracted

from the frozen

section

RIN = 9.5

Storage and RNA Integrity.

RIN of different mouse

tissue RNA samples

before and after 3

year storage

Tissue RIN before

storage

RIN after

storage

Brain 9.2 8.5

Lung 9.5 9.4

Parotid 9.4 8.0

Kidney 9.5 9.1

Bladder 9.4 9.0

Stomach 9.6 9.0

Small intestine 9.4 8.0

Pancreas 7.7 6.6

Bone 9.4 9.2

Liver 9.6 9.3

Esophagus 9.7 9.4

Atria 9.5 9.5

Adrenal 9.7 9.1

Eye 8.7 7.5

RNA stability in storage

depends on tissue type

and initial RNA quality

Storage of Blocks.

Paraffin blocks.

• After completion of sectioning, wrap the block in Parafilm®,

place in air tight bag or food saver bag, seal and return to the

storage.

• Store blocks at RT or +4ºC for DNA and at -20ºC for RNA

applications.

OCT (frozen) blocks.

•Store blocks at -80ºC at the back of the freezer.

•Handle and transfer blocks to -80ºC storage on dry ice.

•Minimize block time inside the cryostat during cutting.

Storage of Slides

Paraffin section of mouse embryo

-20ºC RT

Cut slides for molecular applications right before use for the best results.

Store OCT slides at -80 C at the back of the freezer.

Conclusions

• Treat LCM sample as a genomic sample.

• Take care of LCM sample molecular integrity

at all steps of sample collection and

preparation.

• QC sample preparation for the best possible

result.

• Think- success!

Acknowledgements

NIEHS

Dr. Robert Sills

Julie Foley

SAIC, NCI Frederick

Dr. Serguei Kozlov

Dr. Keith Collins

Staff of the Pathology-Histotechnology

Laboratory

Laser capture microdissection=molecular analysis of ... Genomic Sampling... · microtomy and cryomicrotomy. • RNAse-free conditions: - Wipe everything (including brushes, inside

Documents