The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.
The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.
The prostate gland is a heterogeneous tissue composed of secretory epithelium arranged in glandular acini surrounded by fibromuscular stroma composed primarily of smooth muscle1. The epithelial compartment is comprised of five different but organized cell types: basal cells, secretory cells, neuroendocrine cells, transit amplifying cells and stem cells2. In prostate cancer (PCa), which arises from the luminal epithelial cells, the growth of the adenocarcinoma causes an evident progressive decline of the stromal compartment3. For these reasons, tissue specimens will have distinct differences in the proportion of stromal and epithelial cell type based on the extent of PCa. These differences can lead to biased assumptions of the gene expression data acquired from whole tissues without regard to microdissection of the desired cell type. Therefore, to remove this bias it is essential to separate cell types prior to RNA extraction and gene expression analysis.
Macrodissection or microdissection can be used to physically separate well-characterized epithelial areas from the surrounding stroma4-6. Macrodissection is typically done with a razor blade under a dissecting microscope and works well for separating large PCa nodules from stroma, but is not capable of removing benign epithelium from surrounding stroma (see example of benign prostate histology in Figure 1). Microdissection with a laser (LCM) is significantly more labor intensive than macrodissection, but can very accurately dissect benign epithelium4.
Recent publications from our lab have shown that RNA can be successfully extracted by LCM from either formalin-fixed paraffin-embedded (FFPE) biopsies or frozen tissue4,7-9. The major challenges in LCM-RNA extraction are 1) to accurately dissect the desired areas of the tissue, and 2) to preserve RNA integrity during the LCM and isolation process4,10. RNA isolated from pure cell populations can be used for gene expression analysis by several methods including reverse-transcription quantitative PCR (RT-qPCR)7,8, microarray11, and deep -sequencing12-14.
The goal of this protocol is to isolate total RNA from LCM prostatic epithelium from frozen tissue for downstream gene expression analyses.
All human tissues used for these experiments were acquired via an Institutional Review Board approved protocol and/or exemption at University of Illinois at Chicago.
1. Section Fresh Frozen Prostate onto PEN-slide and onto Charged Glass Slide
2. Hematoxylin and Eosin-Y (H&E) Stain for Histological Mark-up
Note: This if for the tissue on the charged glass slide and NOT the PEN-frame slide for LCM.
3. Toluidine Blue Staining of PEN-frame Slides
Note: This is done the SAME DAY as the LCM. Use depcH2O water for all solutions. Complete all steps in RNase-free area. All coplins must be cleaned with RNase-decontaminating solution.
4. Laser-capture Micro-dissection (LCM)
5. Total RNA Isolation with a Filter-based Kit and Quality Analysis
6. Gene Expression Analysis
Note: Gene expression of long RNA species (like mRNAs and lncRNAs) is shown in Step 6.1 and short RNA species (like microRNAs) is shown in step 6.2. Different kits are available for RT-qPCR and the amount of RNA required varies by kit (as little as 10 ng). RNA sequencing analysis is described in 6.3.
In a previous study we demonstrated the use of LCM to collect epithelial and stromal tissues to compare expression profiling by RT-qPCR of mRNA and microRNAs from frozen and FFPE prostate tissues from the same patient4. LCM is time consuming, particularly if large amount of RNA is to be collected for next-generation sequencing analysis. Therefore, it is crucial to keep the working space and tools RNase free. It is recommended to examine quality and cell-specificity controls in the RNA collected (discussed in more detail in the next paragraph). Figure 1 shows a stained prostate section on a PEN-frame slide under the microscope before and after laser-capturing benign epithelium.
Once the sample is collected and the RNA is carefully extracted, it is important to assess RNA quality and quantity as shown in Table 1. It is not uncommon to observe low 260/280 nm ratios. Concentrating the RNA will improve the 260/280 nm ratios, but it may also cause loss of small RNA species. In addition to quality controls, it is crucial to examine cell type-specific controls to confirm the specificity of the laser-captured area of the tissue (Figure 1B–C). For example, in Figure 1B the expression of AMACR gene was measured to confirm prostate cancer epithelial tissue compared to normal epithelial tissue,. In Figure 1C the expression of NKX3.1 confirmed the absence of stroma in the LCM-collected sample. The quality of the RNA in the specimen can also be compareed to RNA of known, good quality RNA. RT-qPCR Ct values for RNAs isolated from LCM-collected- tissue are in the same range as RNA collected from cultured primary epithelial cells, confirming quality of long and small RNA extracted (Table 2). Lastly, Table 3 shows that this RNA is of sufficient quality for next-generation RNA sequencing.
Figure 1: Collection of benign epithelium, prostate cancer epithelium and stroma from human prostate by laser-capture micro-dissection. (A) prostate biopsy stained with toluidine blue before and after LCM-collection of the benign epithelium. (B–C) Tissues collected by LCM from 45 patients express appropriate gene markers of cellular identity7. (B) AMACR is a marker of prostate cancer and is only detectable in cancer tissues. (C) NKX3.1 is a marker of epithelium and is not detectable in stromal tissues. This figure is modified from Giangreco et al., JSBMB 20157. Please click here to view a larger version of this figure.
Table 1: RNA quality and quantity from LCM-collected prostate tissue.
Table 2: RT-qPCR from LCM collected Prostate benign epithelium.
Table 3: RNAseq from LCM-collected prostate tissue.
Gene expression profiling from human specimens can be challenging, not only for the quality or quantity of tissue available, but also for the various histological entities present in a given tissue specimen. This is particularly challenging in the prostate in which benign tissues are largely stromal tissues and areas of cancer are devoid of stroma. LCM facilitates physical separation of prostatic stroma and epithelium RNA for a more accurate signature of the two different cell types (Figure 1A). In comparison to macrodissection or whole tissue processing, LCM can separate cell compartments in benign tissues, but is significantly more costly and labor intensive.
As in any technique there are possible pitfalls. For example, RNA quality can be partially degraded during initial specimen procurement or during the LCM and extraction procedure. In the first case, gene expression can be achieved measuring different short amplicons by qPCR. In the second case, scrupulous RNase free treatment of all the tools and material should be taken to prevent RNA degradation, as well as a careful handling and processing of the tissue sections. Furthermore, limiting the time spent on one slide to less than 90 min maintains RNA quality during the collection process. The biggest limitation of the LCM technique is the access to an LCM instrument and is the labor required to do the LCM.
There are several types of laser-capture microdissection microscopes available. This protocol describes use of an LCM that uses the laser to circumscribe the areas, which then falls in to a collection cap below. This type of LCM is ideal for separation of epithelium and stroma in prostate tissue which contains discrete multicellular areas to collect, but this type of LCM is not suitable for isolation single individual cells from a tissue. For example, this protocol should not be used to collect resident macrophages, neuroendocrine cells or infiltrating lymphocytes, which are often present as single cells. There are other LCM instruments and methods that utilize a laser to “shoot” single cells from tissue onto a membrane cap, which facilitates isolation of these other cell types.
Thorough quality control and characterization of the RNA cannot be overlooked. Absorbance at 260 nm is suitable to quantify RNA for RT-qPCR studies (Table 1), but for RNA-sequencing a dye-based method more accurately quantifies the RNA. Another important issue in RT-qPCR analysis is to avoid potential bias from residual DNA contamination. This can be accomplished by using primers that span introns. There is also high patient heterogeneity in housekeeper gene expression, therefore use of multiple housekeeper genes is suggested and we typically use three to five4,7-9 (Figure 1B–C and Table 2).
The choice of gene expression profiling method is not only based on the quantity of RNA collected but also on the kind of data that one wishes to acquire and compare. Regular RT-qPCR is more sensitive than next-generation deep sequencing, and requires a relatively small amount of RNA thus decreasing the LCM procedure time4. Next-generation RNA sequencing is an effective technique to quantify the expression both for short and long RNA species. Next-generation sequencing also allows discovery of novel RNA species13. Fortunately, the cost of next-generation sequencing is declining.
In summary, this protocol enables RNA isolation of homogeneous populations of epithelial or stromal cells from frozen prostate tissue. The RNA can be used for numerous downstream analysis techniques to provide a tool for accurate cell type-specific gene expression in the benign and diseased prostate. This type of data contributes to knowledge and understanding of how RNA expression differs in disease pathology.
The authors have nothing to disclose.
We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).
RNase-AWAY | MBP | 7005-11 | |
PEN-membrane 4,0 mm slides | Leica | 11600289 | |
Glass slides, Superfrost Plus | FisherBrand | 12-550-15 | |
Ethanol 200 proof | Decon labs. | 2701 | |
DEPC (diethyl pyrocarbonate) | Sigma | D-5758 | |
Cryostat | Leica | CM3050 | |
Coplins (Staining jar) | IHCWORLD | M900-12 | |
Coplins (Staining rack) | IHCWORLD | M905-12 | |
Aperio ScanScope | Aperio(Leica) | ScanScope® CS | |
Toluidine Blue | Fluka | 89640-5G | |
Laser Microdissection System | Leica | LMD7000 | |
0.5 mL Thin-walled Tubes for LCM | Thermo Scientific | AB-0350 | |
RNAqueous®-Micro Total RNA Isolation Kit | Ambion | AM1931 | Thermo Fisher Scientific Brand |
NanoDrop | Thermo Scientific | ND-1000 | |
Qubit 2.0 Fluorometer | Life Technologies | Q32866 | Thermo Fisher Scientific Brand |
High-Capacity cDNA Reverse Transcription Kit | Applied Biosystems | 4368814 | Thermo Fisher Scientific Brand |
Universal cDNA Synthesis Kit II, 8-64 rxns | Exiqon | 203301 | |
TaqMan microRNA RT kit | Applied Biosystems | 4366597 | Thermo Fisher Scientific Brand |
Hematoxylin stain | Ricca Chemical Company | 3536-16 | |
Eosin-Y | Richard Allan Scientific | 7111 |