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Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies
© Kulkarni et al.; licensee BioMed Central Ltd. 2013
- Received: 5 April 2013
- Accepted: 9 October 2013
- Published: 26 October 2013
The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays.
Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection.
The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded.
The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms.
Human donor eyes are important source of tissue for research into pathogenesis, treatment and prevention of disease [1, 2]. Obtaining samples of pure cell population is crucial for transcriptomics studies, as contamination with other adjacent cell populations leads to increased biological noise in the microarray data, which interferes with the detection signals from genes of interest in the tissue samples . Laser microdissection (LMD) allows precise collection of cell populations of interest from heterogeneous tissue and preserves the purity and integrity of RNA samples. This is crucial for the accuracy of the microarray results to determine significant differences in gene expression between treatment and control groups .
An oligonucleotide microarray was successfully performed on laser microdissected tissue samples by Ohyama H et al. in 2000 . A study on laser microdissected breast tissue samples by Cowherd et al. in 2004 showed that the technical variability introduced by amplification and hybridisation experiments is smaller than the actual biological variation for different types of breast cancer tissues demonstrated with differential gene expression in these samples . Samples for transcriptional profiling studies on epidermal basal cells, endometriosis tissue, and osteocytes were successfully collected with “Positioning and Ablation in Laser Microdissection” (PALM®) microbeam systems (PALM Microlaser Technologies GmbH) . This technique grew in popularity as it allowed precise dissection of tissues under direct visualisation by a non-contact technique.
Principle of LMD technology
This article describes in details, the methodology of obtaining laser microdissected RNA samples from the human OS epithelial regions such as the cornea, limbus, conjunctiva and Limbal Epithelial Crypt (LEC) for transcriptional profiling with consideration of factors influencing the quantity and quality of these samples such as staining procedure, duration of LMD and the area of tissue dissected.
Materials and supplies
15 mm trephine
2% v/v povidone-iodine solution
Optimum temperature compound (OCT, Emitech Ltd, East Sussex, England)
Jung CM 1900 cryostat (Leica Microsystems Ltd., Milton Keynes, UK)
PALM® collection tube (PALM, Bernried, Germany; Product code 1440–1000)
70% v/v ethanol
RNase free 0.1% w/v Toluidine Blue
0.1% diethyl pyrocarbonate (DEPC) treated water
1% β-Mercaptoethanol (Sigma Aldrich, Gmbh, Germany)
RNeasy Micro kit (Qiagen, West Sussex, UK)
PALM Membrane slides (PALM # 1440–1000)
LPC Microfuge tubes, 2 mm rim, 500 μl (PALM # 1440–0200)
Latex disposable gloves
11 mm scalpel blade
Cryospray (Cell Path Ltd, UK)
Ambion® Message Amp™ II aRNA Amplification Kit
NuGen WT-Ovation™ Pico RNA Amplification System
Ethical approval and retrieval of the cadaver eyes
This research project was approved by the Nottingham Research Ethics Committee and the Research and Development department of the Nottingham University Hospitals National Health Service Trust. Donor eyes not used for transplantation but also consented for research by the relatives of the deceased, and eyes harvested for research purpose only were included in the study. Retrieval of the cadaver eyes was performed with conventional techniques within 24 to 36 hours of death to maintain the cellular RNA viability of the OS epithelium. Processing of the tissues following prolonged post mortem interval could affect the quality of the samples due to degradation of tissue RNA and drying of the OS epithelium following atmospheric exposure. Quality of the OS epithelium in the donor eyes was assessed with the dissecting microscope prior to the tissue preservation.
The inclusion criteria for donor eyes was: i) Donor age between 20 to 70 years; ii) Donors of either sex; and iii) Eyes with healthy OS epithelium. Donor eyes selected for research were logged in an eye tissue database for use in the study.
Ocular tissue processing and preservation
To prevent RNase contamination of the donor tissue the following precautions were taken; cleaning bench tops and lab equipment with Trigene and Ethanol (EtOH), use of disposable gloves, sterile disposable instruments, petridishes and blades. Whole eyeball supported on an eye stand was cleaned by immersing in 2% v/v povidone-iodine solution for two mins, followed by a sterile PBS wash. A corneoscleral button with 3 mm frill of conjunctiva surrounding the limbus, to include palisades of Vogt and LEC was dissected from the cadaver eye with a 15 mm trephine . The corneoscleral button was placed in a petridish with PBS to prevent drying of the OS epithelium and cut radially into eight triangular segments with a disposable scalpel blade. Handmade aluminum foil cups of approximately 20 mm diameter were filled with an embedding medium optimum temperature compound (OCT, Emitech Ltd, East Sussex, England). Each triangular segment of corneoscleral button was oriented in the OCT compound, in such a way that one of the long edges of the triangle was parallel to the surface of the OCT medium and short edge perpendicular to the superficial edge. Frozen tissue blocks were prepared by immersing the OCT embedded tissue in Isopentane bath pre-chilled (frozen) in liquid nitrogen. Frozen blocks were stored at −80°C till further use.
Cryosectioning was performed with the Jung CM 1900 cryostat (Leica Microsystems Ltd., Milton Keynes, UK). Collection and processing of the tissue including RNA extraction was performed under RNase free conditions. Thorough cleaning of the cryostat chamber was performed with absolute EtOH and acetone prior to use for prevention of tissue contamination. The tissue sections of interest were taken on PALM slides and fixed for 5 mins in 70% EtOH pre-chilled in the cryostat. The slides were then air-dried briskly and placed in a pre-chilled slide box for storage at −80°C for LMD.
Pre laser microdissection processing of tissue sections
Immediately, prior to LMD, cryosections on the PALM slides were thawed on ice and stained with RNase free 0.1% w/v Toluidine Blue dye for 30 seconds, by pipetting approximately 200 μl of the dye over each of the tissue section. The stained slides were then rinsed twice in 0.1% diethyl pyrocarbonate (DEPC) treated water and air dried for 5 mins.
Laser microdissection (LMD)
The laser parameters such as the focal diameter of the beam, magnification and numerical aperture of the applied objective lens were adjusted for efficient results. The length of the OS epithelial region was divided perpendicularly into smaller segments with the cut function of the laser (Figure 2A, D and J). The cuts were extended lengthwise for complete dissection of the area of interest. Following this, the whole tissue fragment was catapulted in the cap of the collection tube with RoboLPC function, which is a combination of LMD and pressure catapulting (Figure 2B, E, H and K). In the collection tube the tissue segment sticks to the thermostable membrane at the base of the cap. In case a segment failed to eject into the collection tube, the misdirected piece of tissue was catapulted into the collection tube with the auto Laser Pressure Catapulting function (LPC). On completion of LMD, the collection tube was moved directly over the objective lens using the joystick to check for the catapulted specimens in the collection cap (Figure 2C, F, I and L). Thereafter the collection tube was removed and 300 μl of RNA lysis buffer (RLT) (RNeasy Micro kit, Qiagen) containing 1% β-Mercaptoethanol (Sigma Aldrich, Gmbh, Germany) was added to the tube, the collection tube cap was closed, and the tube sealed with parafilm to prevent contamination during storage. The tube was inverted, briefly vortexed and incubated at room temperature (RT) for 20 mins, to lyse the tissue cells sticking to the roof of the cap and release the RNA. Subsequently the RLT tubes were stored at −80°C until further use.
Histological identification of OS epithelium
Total RNA extraction of LMD tissue was performed with RNeasy Micro kit, according to the manufacturer’s protocols (Qiagen, West Sussex, UK). Each sample was made up to 350 μl with RLT buffer (activated with 10 μl of β-Mercaptoethanol per 1 ml of buffer RLT).
This section mainly presents the effect of optimisation of LMD technique with consideration to cryopreservation of the tissue, thickness of the cryosections taken on PALM slides, fixation technique and tissue staining.
Optimisation of sample processing for LMD
Optimisation of the sample preparation and LMD technique was crucial for efficient time and cost management of the project. Practically all the steps of the LMD were optimised, and their effectiveness observed. The findings are described below.
Freezing of tissue blocks
The tissue processing for LMD was performed taking care to preserve the RNA integrity of the processed tissue. On histological examination, it was noted that tissue sections of tissue blocks snap frozen in liquid Nitrogen developed cracks or freeze fractures. This compromised the tissue quality due to which the tissue had to be discarded. To avoid this problem controlled gradual freezing of the tissue was performed in Isopentane pre-chilled in liquid Nitrogen.
Thickness of the cryosections
Following optimisation of tissue thickness, cryosections of 6 μm thickness were found to be effective for LMD. It was noted that thin sections (< 6 μm) folded easily or tore while transferring on to the slides. This made the OS epithelium inaccessible to LMD and the affected tissue sections had to be discarded. On the other hand, thick sections (> 6 μm) were easily lost and fell off the slide during fixation and washes. It was also noted that during LMD the thick sections did not catapult effectively into the collection cap and got lost or misdirected over the surrounding tissue.
Consistent fixation of tissue sections to the slides and preservation of morphology of OS epithelial regions was achieved with 70% EtOH pre-chilled at −20°C.
Number of sections on each slide
The influence of the number of tissue sections on the PALM slide was investigated using 1–6 sections per slide. It was noted that, more than 5 sections caused crowding of the tissue sections on the slide leading to overlapping. This resulted in loss of tissue for LMD and less RNA yield. Taking 1 to 2 sections per slide did not have an adverse effect on the RNA quality; but this was not cost effective. However, the cost effectiveness was maximised by taking 3 to 4 tissue sections on the PALM slide.
Processing of the tissue sections for LMD
Pre LMD tissue section staining was performed with RNase free toluidine blue dye in combination with DEPC treated water (RNase free) for better identification of tissue morphology without compromising RNA integrity . To maintain the integrity of the tissue RNA, LMD of the toluidine stained tissue sections was performed within 3 hours of the staining.
Optimisation of the laser function for LMD
Details of samples processed for spotted microarray showing LMD area, corresponding concentration, of unamplified, amplified and labelled RNA samples with FOI values
ch1: source name
LMD area, μm2
RNA Conc pg/μl
Conc of amplified RNA ng/μl
Labelled extract ng/μl
FOI of Cy5 dye
SC-CO398 LEC Stroma
SC-CO399 LEC Stroma
SC-CO404 LEC Stroma
SC-CO418 LEC Stroma
Ch2: source name Corneal + Conj epithelium
RNA conc μg/μl
2nd Round amplified RNA ng/μl
Labelled extract ng/μl
FOI of Cy3 dye
LEC had the highest density of cells and yielded a good concentration of RNA inspite of variations in number and sizes of LEC laser microdissected. However, the LEC stroma had sparse distribution of cells hence large areas from this region were laser microdissected to obtain sufficient concentrations of RNA.
Optimisation of LMD tissue for Oligonucleotide microarray experiments
Four biological replicates from four pairs of eyes were processed for each OS epithelial region including the LEC, limbus, cornea, conjunctiva and LEC stroma. Table 1, shows unamplified, amplified RNA concentration values including the labelled probes of all the LMD samples processed in this microarray study. It also includes the concentration values of the reference probes (SP) and the concentrations of labelled extracts of samples and the Standard Probes with the Frequency of Incorporation (FOI) of the dyes in these samples. The labelled probes of the samples were matched to the Standard Probe with similar FOI to generate hybridised probes.
Validation of RNA viability with semi quantitative PCR
Optimisation of LMD tissue for Gene 1.0 ST array
LMD samples used in Gene ST 1.0 array experiments
LMD area μm2
RNA conc (ng/μl)
Optimisation of LMD tissue for real time PCR
Five cadaveric eyes were processed for real time PCR experiments to validate the oligonucleotide microarray studies. The LMD area ranged from 85,831 μm2 to 138,868 μm2. The OS region samples were processed in triplicates to demonstrate replicable results.
Successful isolation of the tissue or cells by LMD involves appropriate collection or harvesting of the donor, with contamination free sectioning, processing and storage of the samples at −80°C to preserve the RNA quality prior to LMD.
Mechanical isolation of the limbal epithelium and the LEC without contamination from adjacent tissue is technically challenging, as the limbus is a narrow one mm transient zone between the cornea and conjunctiva and LEC is a deep seated structure arising from the under surface of limbus extending into the underlying limbal stroma. Similar observation regarding the limbus was previously made in a molecular study using LMD tissue . LMD offers a viable and effective option to isolate cell populations of interest from heterogeneous tissue thus preserving the purity and integrity of the RNA samples for further molecular studies .
Pinzani et al. in 2006 had demonstrated that directly immersing the tissue in liquid nitrogen for cryopreservation disrupts the tissue morphology due to the formation of ice crystals leading to freeze fractures in the tissue. The authors have further suggested that controlled freezing of the tissue prevents the development of freeze fractures in the tissue and have demonstrated extraction of good quality RNA and DNA from the controlled frozen tissue .
The authors have further noted that tissue sections of 5-8 μm thickness are ideal for microscopic resolution due to monolayer cell thickness of these tissue sections. A similar protocol study on LMD tissues have reported effective RNA extraction of LMD tissue processed from 8 μm tissue cryosections .
In this study, cryosections of 6 μm thickness was noted to improve the histology quality of sections and visualisation of the LEC.
In a study on LMD tissue, Kerman et al., had demonstrated a relationship between sectioning strategy and the RNA quality. They had observed that mounting 1–2 sections on the slide had minimal influence on the RNA quality; however, the RNA quality dropped when four or more sections were mounted per slide. The authors had noted that mounting more sections on a slide caused longer exposure of the sections to the room temperature while being processed, leading to uncontrolled RNase activity and hence RNA degradation during this period. Based on the abovementioned observation and our own experience, we had limited maximum of 4 sections on each PALM slide. This not only improved the cost effectiveness but also maintained the RNA quality of the processed tissue at the same time.
Furthermore, it was noted that post-staining handling of the slides for LMD for up to 3 hours did not significantly influence the quality of RNA. However, this processing time should not be exceeded; as such delays adversely affected the RNA quality . Based on observations of this study all the toluidine stained tissue sections were laser microdissected within 3 hours of processing.
A study on LMD process has established better preservation of tissue morphology following fixation with 70% acetone at −20°C compared to 70% EtOH however, the RNA recovery was similar in both methods. For optimal results, the authors have suggested that tissue sectioning should be carried out at −20°C and have recommended use of membrane coated slides for excellent tissue capture during LMD . Similarly, in our study, soon after the tissue sections were taken on the PALM slide the slides were retained in the cryostat chamber at −20°C until fixed in pre-chilled (−20°C) 70% v/v EtOH to prevent RNase activity. We had noted good preservation of morphology of OS epithelial regions following fixation in 70% EtOH during optimisation process.
A LMD study had demonstrated the effective staining of the tissue sections with toluidine blue, a nuclear dye resulting in preservation of RNA integrity and reliable amplification of cDNA with Taqman real-time PCR . Similarly studies on the staining procedures for LMD sections, have suggested the use of RNase free aqueous staining solutions for preparation of the toluidine dye and washes to protect RNA integrity . The authors of the latter study have recommended processing of the LMD tissue under sterile conditions and storage at −80°C in the lysis buffer until further use. During RNA extraction, they have advised DNase treatment of the samples to prevent genomic DNase contamination .
Comparison of RNA extraction of the LMD tissue with Qiagen RNeasy Micro kit (Valencia, CA) and Trizol RNA isolation reagent (Life Technologies, Invitrogen, Carlsbad, CA) have demonstrated efficient RNA extraction from LMD frozen tissue with Qiagen RNeasy Micro kit (Valencia, CA) .
In this study, dissection of variable amounts of epithelial tissues was unavoidable due to factors such as limited availability of LEC samples and sparse cellular density of LEC stroma. This limitation was dealt with by scrutinising wet unstained 6 μm thick serially cut cryosections involving 360° of the limbus, for LEC and including all the LEC sections for LMD to maximise the tissue availability. For LEC stroma samples large area of tissue was laser microdissected to obtain adequate RNA from this region.
During LMD due to the absence of cover slip over the tissue section visualisation of the morphology of the tissue becomes difficult therefore to facilitate identification of the morphology the tissue sections needs to be stained this can affect RNA viability.
LMD products in lysis buffer and RNA samples are unsuitable for long term storage.
Molecular study involving pure cell populations might not consider interaction between the cell population studied and surrounding structures such as supporting cells and the extracellular matrix. This may result in lack of information regarding cell to cell or cell to extracellular matrix signalling.
Certain downstream molecular procedures such as microarray experiments and proteomics may require large amount of samples, which may not be feasible to collect with LMD.
LMD needs to be performed in a limited period of time, as degradation of tissue samples may be an issue due to exposure during prolonged laser microdissection.
The amount of tissue sample needed is also determined by amount of target molecule per cell and number of replicates required.
Troubleshooting in Laser microdissection of OS regions
Contamination of samples
Contamination of contact surfaces such as cryostat blade, staining solutions and tissue slides with RNase
Use gloves and follow sterile method of sample preparation. Wipe down the work surfaces and instruments with Trigene, alcohol and also possibly with RNase zap sprays. Change cryostat blade after every use. Use fresh staining solutions which are prepared in RNase free solutions for tissue staining. Use sterile PALM slides and restrict the number of tissue sections per slide to 3–4. Don’t allow the tissue sections to thaw unnecessarily as it activates the RNases in the tissue.
Unsatisfactory tissue staining resulting in difficulty in identification of tissue morphology in microscope.
Using too diluted staining solution. Incubation of tissue sections in staining solution inadequately. Thick tissue sections.
Use predetermined and optimised concentration of the staining solutions. Follow the recommended staining procedure. Increase incubation for staining.
Inadequate laser microdissection of tissue section
Thick tissue sections, tissue section placed near the margins of the slide resulting in tissue section or part of it not in the laser optical plane.
Ensure adequate dehydration of tissue section following staining procedure. This could be noted by checking for watermarks on the membrane of the PALM slide. Place tissue sections in the centre of the membrane oriented parallel with each other to prevent overlapping and folding of the sections.
Failure or misdirection of cut tissue segment to catapult in the collection tube
Tissue section not dehydrated satisfactorily.
Prior to beginning of the LMD optimise the laser settings to facilitate adequate cutting without singing or burning of the surrounding tissue seen as black frill around the cut edges.
Laser used is either out of focus or of inadequate power.
Small tissue segments are effectively catapulted in the collection tube
Large area of tissue segment dissected
Adequate dehydration of the slide could be ensured by incubating the slide in warm air incubator for approximately 5 minutes or fixing the stained slide in the ethanol bath
Tissue section not adequately dehydrated
Following LMD scan the slide surface under microscope to detect any misdirected pieces which could be redirected in collection tube by realigning the collection tube over the tissue segment and recatapultion of tissue segment with LPC laser function
Inadequate laser power
Check the collection tube cap for number of tissue segments catapulted in comparison to actual LMD segments
Inadequate RNA quality and quantity
RLT buffer leaks from the collection tube
Pooling of the RNA samples from same biological replicate
Contamination of RNA samples while processing
Variable amounts of RNA
LMD is an effective non-contact method of collection of tissue of interest from heterogeneous tissue samples for downstream molecular experiments. Optimisation of tissue collection, processing and LMD technique was found to be crucial to maximise the effectiveness of LMD process for generation of viable RNA samples of sufficient concentration and quality. We have demonstrated that picogram to nanogram amounts of laser microdissected RNA samples generated from insitu OS epithelial regions of human donor eyes can be successfully used for downstream molecular analysis such as microarrays and real time PCR.
I am grateful to the Royal College of Surgeons of Edinburgh, UK for providing financial support to this project. I wish to thank Dr Patrick Tighe, Dr Matthew Arno, Toshana Foster, and Estibaliz.Aldecoa-Otalora_Astarloa for their technical support with microarray experiments
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