Isolation Of High-quality RNA Research Articles

Single Cell Type Specific RNA Isolation and Gene Expression Analysis in Rice Using Laser Capture Microdissection (LCM)-Based Method.

The success of single cell type-specific gene expression or functional study largely depends onthe efficient isolation of high-quality RNA from them. Laser capture microdissection (LCM) is an efficient technique that allows accessing and dissecting out a specific individual cell or cell type from a microscopic heterogeneous tissue in a minimally disruptive way. Here, we describe an efficient and inexpensive LCM-based method for theextraction of RNAs with high yield and integrity from laser-microdissected mesophyll and bundle sheath cells of rice leaf. Theintegrity of isolated RNA is assessed with bioanalyzer analysis, and the presence of mRNAof aspecific gene is validated through RT-PCR. This RNA could further be used for uncovering single cell type-specific gene expression signature using next-generation transcriptome sequence or through regular RT-PCR.

RNA Isolation from Articular Cartilage Tissue.

Isolation of high-quality RNA directly from tissues is desirable to obtain precise information of in vivo gene expression profiles in cells embedded within their extracellular matrix (ECM). It is well known that purification of RNA from cartilage tissues is particularly challenging due to low cell (chondrocyte) content and its dense ECM rich in large negatively charged proteoglycans that can copurify with RNA. Older methodologies to purify RNA from cartilage involved the use of concentrated denaturing solutions containing guanidinium isothiocyanate followed by ultracentrifugation in cesium trifluoroacetate. Such ultracentrifugation approaches are rarely used now since the emergence of more user-friendly mini spin column chromatography kits. For this chapter, we tested and compared three methods to isolate RNA from immature murine articular (femoral head) cartilage and found that the combination of TRIzol® reagent and spin column chromatography (Norgen Total RNA Purification Kit) was the best approach to generate higher quality RNA. Here, the average RNA Integrity Number (RIN), as determined by Bioanalyzer technology, was 7.1. We then applied this method to attempt to isolate RNA directly from human articular cartilage harvested from three osteoarthritic (OA) knee joint specimens. As expected, the concentration and quality of RNA obtained differed between samples. However, from one specimen, we were able to isolate approximately 3μg of total RNA (including small noncoding RNAs) from 100mg of human OA cartilage with a RIN=7.9. Despite the patient-to-patient variabilities that are known to exist between cartilage specimens from OA joints, we have demonstrated that it is possible to obtain reasonably high levels of RNA from human OA articular cartilage at a quality suitable for downstream analyses including microarray and RNA-Seq. A detailed description of our preferred RNA purification methodology, which can be used to isolate RNA from human, bovine, or rodent cartilage tissue, is provided in this chapter.

An easy and robust method for the isolation of high quality RNA from coconut tissues

Background: Coconut tissues consist of a complex network of polysaccharides, proteins, polyphenols, and lipids that can bind to nucleic acids and pose difficulty in isolation. Certainly, a vigorous method is required to isolate high quality and quantity of RNA from such tissues for the purpose of downstream experiments. In this paper, we discuss a newly developed method for the Isolation of RNA from Complex Matrices (IRCM) method from coconut tissues. Results: The method is robust, cheap, and efficient for the extraction of quality RNA in high quantities from the solid endosperm of stored and fresh coconut (150 μg/g FW with A260/280 = 1.89 and 247.5 μg/g FW with A260/280 = 1.91), coconut apple (263.8 μg/g FW with A260/280 = 1.97), and coconut bud (1052.5 μg/g FW with A260/280 = 2.00). The other well established methods, such as Method of RNA Isolation from Palm (MRIP), Cetyl Trimethyl Ammonium Bromide (CTAB), TRIZOL, and RNA plant kit failed to isolate quality RNA in appreciable quantities from the coconut tissues. Furthermore, the resultant RNA performed well in the downstream experiment, that is, RT-PCR for the production and amplification of cDNA. Conclusions: From the study, we concluded that the present method will play a vital role in the extraction of high quality RNA from complex matrices in a short time. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

Two-step method for isolation of high-quality RNA from stored seeds of maize rich in starch.

A modified SDS-Trizol method was optimized for isolation of total RNA from the stored maize seeds at regular interval of one month for 4 months. Use of SDS extraction buffer before the use of Trizol reduced the co-precipitation problem associated with high carbohydrate content in the seed. Recorded mean RNA yield from seeds across the storage intervals was 978.6 ± 65.46ng/µl. Average spectrophotometric values (A 260/280) of isolated RNA varied from 1.974 ± 0.033 to 1.998 ± 0.022. Attempts to isolate RNA from green leaves using Trizol method also ensured comparable quality and quantity of the isolated RNA. RNA yield from fresh leaves was recorded 1008.2 ± 77.088ng/µl which is slightly higher than the mean RNA yield from seeds across months. Observed mean A 260/280 values of isolated RNA were 1.984 ± 0.030. DNase treatment further improved the A 260/280 ratio in both seeds (2.003 ± 0.006) and leaves (2.012 ± 0.037). High quality and quantity along with integrity of the isolated RNA was ensured through downstream analysis after RNA extraction such as first-strand cDNA synthesis and normal PCR. Extraction of RNA from the stored seeds using modified SDS-based Trizol method and from fresh leaves using Trizol method opened new possibility of understanding role of key genes involving developmental steps especially in the stored seeds.

Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells.

Previous studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further in silico analysis of transcriptional data suggested a critical role for STAT5 transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of STAT5 was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with STAT5 signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.

Laser Microdissection of Cells and Isolation of High-Quality RNA After Cryosectioning.

Laser capture microdissection (LCM) has become a powerful technique that allows analyzing gene expression in specific target cells from complex tissues. Widely used in animal research, still few studies on plants have been carried out. We have applied this technique to the plant-nematode interaction by isolating feeding cells (giant cells; GCs) immersed inside complex swelled root structures (galls) induced by root-knot nematodes. For this purpose, a protocol that combines good morphology preservation with RNA integrity maintenance was developed, and successfully applied to Arabidopsis and tomato galls. Specifically, early developing GCs at 3 and 7days post-infection (dpi) were analyzed; RNA from LCM GCs was amplified and used successfully for microarray assays.

Double lysis: an integrative time-saving method yielding high-quality RNA from strawberry

BackgroundThe isolation of high-quality RNA from strawberry leaves and fruits is notoriously cumbersome. Both tissues are extremely rich in active secondary metabolites, as phenolics and pigments that inevitably perturb the isolation of RNA. Many protocols have been developed to address this problem. However, they are either costly, or time-consuming, in particular for high number of many plant samples. We describe here a new method with an easy-to-handle approach to obtain high-quality RNA from strawberry leaves and fruits. The method, referred to as double lysis, uses a novel combination of CTAB and Trizol protocols. ResultsCompared to conventional Trizol-dependent protocols, either used individually, or in a commercial spin-column kit, the new method yields RNA at lower costs, but of significantly improved quality. The RNA obtained by double lysis was very pure as indicated by 260/280 ratios of 2.06 (leaves) and 2.07 (fruits), while 260/230 ratio was 2.07 and 1.75, respectively. Also visually, RNA sediments from double lysis showed a white color, indicative of efficient elimination of polyphenolics and pigments. In contrast, traditional Trizol method produced reddish precipitates. The purity of RNA isolated by double lysis enabled successful removal of genomic DNA and, thus, allowed for more efficient cDNA synthesis and RT-PCR. In addition, the suggested method is able to yield RNA with fully preserved integrity from strawberry leaves, fruits in addition to petals and roots. ConclusionDouble lysis is a new RNA isolation protocol developed through the integrative coupling of CTAB and Trizol reagents. The method is cost-effective, robust, time-saving, and can cope even with recalcitrant plant tissues with high contents of phenolics and pigments, such as strawberry leaves and fruits.

Isolation of high-quality RNA from recalcitrant leaves of variegated and resurrection plants.

Resurrection plant Ramonda serbica is a suitable model to investigate mechanisms of desiccation tolerance, while variegated Pelargonium zonale has been proven to serve as an excellent model for the metabolite allocation between sink tissue and source tissue within the same organ. However, the genomes of these plants are still not sequenced, limiting their application in molecular studies. To investigate the transcript abundance by next-generation sequencing, high-quality RNA input is required. Leaves of both P. zonale and R. serbica are rich in polyphenols that interfere with high-quality RNA extraction by common protocols. Moreover, low water content and high amount of sugars and other osmoprotectants in desiccated R. serbica leaves present the additional challenge in total RNA extraction. Here, we evaluated and compared several already established TRIzol- and CTAB-based protocols aiming to develop the efficient, simple and low-cost methods for the extraction of the satisfactory yield RNA of great purity and integrity, required for the construction of high-quality cDNA libraries. Our results show that the CTAB-based protocol (i.e. CTAB 1b) enabled the extraction of high-quality RNA from photosynthetically active and non-photosynthetically active leaf sectors of P. zonale, with high RIN values. On the other hand, TRIzol-based protocol provided a high RNA yield with low contamination and high RNA integrity even in desiccated leaves of R. serbica. We envisage that the proposed protocol would be suitable for the RNA extractions from other desiccated organs (e.g. seeds, grains, pollen grains).

An improved method for isolation of high-quality RNA from starch-rich wheat grains

An improved method for isolation of high-quality RNA from starch-rich wheat grains

Extraction of high-quality RNA from mouse pancreatic tumors

Extraction of high-quality RNA from pancreatic tumors for sequencing purposes is technically challenging, as the pancreas is an organ rich in ribonucleases. The majority of the established RNA isolation protocols for use with primary pancreatic tissue involve perfusion of RNA stabilizing reagent into the pancreatic tissue to protect RNA integrity before extraction. However, the additional time needed for this procedure can actually lead to further RNA degradation. We optimized a protocol suitable for high quality RNA isolation from mouse pancreatic tumors that is a simple, fast, and inexpensive modification of existing methods, combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction. Through this procedure, the mean RNA Integrity Number value obtained for RNA isolated from pancreatic tumors was 9.0, and was reproducibly suitable for RNAseq and qPCR.•a protocol suitable for high quality RNA isolation from mouse pancreatic tumors as well as normal pancreas•combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction

Techniques for Small Non-Coding RNA Analysis in Seeds of Forest Tree Species.

In recent years, the scientific community has become aware that epigenetic mechanisms play a more important role in gene regulatory networks (GRNs) than was hitherto thought, as accumulating evidence has shown that changes in epigenetics without genetic variation can affect complex traits over multiple generations. Within the epigenetic machinery, small non-coding RNAs (sRNAs, 18-24 nucleotides in length) are evolutionarily conserved RNA molecules that target mRNAs for deregulation or translational repression. They commonly have high-level regulatory functions in GRNs by mediating DNA and/or histone methylation and gene silencing essential for plant developmental programs and adaptability. Local adaptation enables plants to acquire a high fitness by, for example, properly timing developmental transitions to match plant growth stages with organism's favorable seasons. In particular, the seed represents a key evolutionary adaptation of seed plants that facilitates dispersal and reinitiates the development coupled in time with suitable environmental conditions. With the advent of high-throughput sequencing for sRNAs and computational approaches for sRNA detection and categorization, it is now feasible to unravel how sRNAs contribute to the fitness of tree species that can survive hundreds of years (e.g., conifers). Of particular interest is to disentangle the roles of sRNAs from complex genomic information in tree species with intimidating genomic sizes (commonly 20-30Gb in conifers) and abundant nongenic components (e.g., >60% transposable elements). In this chapter, we use seeds of the conifer Picea glauca as a study system to describe the methods and protocols we used or have recently updated, from high-quality RNA isolation to sRNA identification, sequence conservation, abundance comparison, and functional analysis.

High-quality RNA isolation from pigment-rich Dendrobium flowers.

Isolation of high-quality RNA from Dendrobium flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of Dendrobium was optimised. The optimised method yielded high quantities of RNA (10.1-12.9µg/g). Spectrophotometric values of A260/280 in the range of 2.2 to 2.4 and A260/230 values of 2.0 suggested that the isolated RNA was free of polyphenols, polysaccharides, and protein contaminants. RNA integrity numbers determined by microfluidics were in the range of 7.9-8.9 indicative of intact RNA. In the improved method, the addition of 3M NaCl and 3% PVP-10 in the extraction buffer, followed by an incubation period of 45min at 65°C, eliminated most of the polysaccharides, polyphenolic compounds, and denatured protein. Extraction with phenol:chloroform:isoamyl alcohol (125:24:1) effectively removed pigments from the aqueous phase, while the precipitation of RNA with lithium chloride minimised the co-precipitation of protein, DNA, and polysaccharide and resulted in the extraction of high quality of RNA. The suitability of the RNA for downstream processing was confirmed via RT-PCR amplification of Chalcone synthase gene from cDNA prepared from RNA isolated from different developmental stages of the flower of a Dendrobium hybrid. The present method will be highly useful for the isolation of RNA from pigment, polyphenol, and polysaccharide-rich plant tissues.

An optimized protocol for isolation of high-quality RNA through laser capture microdissection of leaf material.

Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low‐melting‐point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high‐quality RNA suitable for down‐stream applications such as quantitative reverse transcriptase–polymerase chain reactions or RNA‐sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.

Effective and economical column-based method for RNA isolation from animal cells.

To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120ng/5 × 106 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.

Improved laser capture microdissection (LCM)-based method for isolation of RNA, including miRNA and expression analysis in woody apple bud meristem.

Isolation of high-quality RNA, including miRNA, from microscopic woody apple bud meristem using laser capture microdissection-based method. It is often challenging to study the expression of microRNAs (miRNAs) or genes in less accessible inner tissues of tree species rich in polyphenols or polysaccharides. Here, we report a laser capture microdissection (LCM)-based method for efficient and cost-effective isolation and expression analysis of miRNAs and genes in the meristem tissueofwoody apple bud. The tissue fixation, processing, infiltration, and sectioning steps were optimized for LCM-based excision and subsequent RNA isolation. Further, we have confirmed that RNA isolated from LCM-derived apple bud meristem contained miRNAs and was of good quantity and quality, sufficient for downstream expression analysis.

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum.

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (≥8) as needed for transcriptional profiling experiments.

QRREM method for the isolation of high-quality RNA from the complex matrices of coconut.

Complex plant tissues vary in hardness, i.e. some are succulent, while others are complex to break. Besides, plant metabolites, such as polysaccharides, proteins, polyphenols and lipids, can greatly interfere with the RNA extraction. So, in order to obtain a high-quality RNA from the complex tissues (like coconut endosperm, coconut apple and coconut leaf bud) rich in secondary metabolites, a robust method is demanded. Several methods (MRIP, CTAB and TRIZOL) have been used previously for the isolation of quality RNA from the coconut tissues, but without any success. The present study will provide with the details of a new method (Quick and Reliable RNA Extraction Method or QRREM), which have efficiently isolated the intact RNA form the complex tissues of coconut compared with CTAB, Trizol and RNA plant. The method has been validated for the isolation of high-quality intact RNA from the other available plant species (Areca/betel nut, mint and spring onion). The method has various advantages over the other methods in terms of time and cost effectiveness. Furthermore, the resulted RNA from various tissues of coconut performed well in the downstream experiments, i.e. reverse transcription and PCR for the production and amplification of cDNA.

Efficient and High-Quality RNA Isolation from Metabolite-Rich Tissues of Stevia rebaudiana, an Important Commercial Crop.

Stevia rebaudiana, a perennial herb native to northeastern Paraguay, has gained immense attention globally over the recent decades due to the natural sweetness of its leaves. Like in most plants, this particular species contains high amount of secondary metabolites, thus rendering the isolation of high quality and quantity RNA extract for molecular applications rather challenging. An effective, high-yield and high-quality RNA isolation protocol for this economically important plant species was devised here based on the cetyltrimethylammonium bromide (CTAB) extraction method, with an additional genomic DNA (gDNA) removal step. DNA and other contaminants that may affect downstream applications were effectively removed. Our results exhibited that RNA samples isolated from the leaves and stems of Stevia rebaudiana using this improvised method are high in integrity and quality with RNA integrity number (RIN) of more than 8 and low in contaminants.

Isolation of photoreceptors from mature, developing, and regenerated zebrafish retinas, and of microglia/macrophages from regenerating zebrafish retinas

This paper describes experimental procedures for the dissociation of retinal cells of the zebrafish (Danio rerio) for subsequent fluorescence-activated cell sorting (FACS) and gene expression studies. Methods for dissociation of zebrafish retinas followed by FACS and RNA isolation were optimized. This methodology was applied to isolate pure sorted samples of rods, long wavelength-sensitive (LWS) cones, medium wavelength-sensitive (MWS; RH2-2) cones, short wavelength-sensitive (SWS2) cones, and UV-sensitive (SWS1) cones from retinas obtained at selective life-history stages of the zebrafish, and for some of these photoreceptors, following retinal regeneration. We also successfully separated lws1-expressing and lws2-expressing LWS cones from fish of a transgenic line in which lws1 is reported with green fluorescence protein (GFP) and lws2 is reported with red fluorescence protein (RFP). Microglia/macrophages were successfully sorted from regenerating retinas (7 days after a cytotoxic lesion) of a transgenic line in which these immune cells express GFP. Electropherograms verified downstream isolation of high-quality RNA from sorted samples. Examples of post-sorting analysis, as well as results of qRT-PCR studies, validated the purity of sorted populations. For example, qRT-PCR samples derived from isolated Rh2-2 cones contained detectable rh2-2 (opn1mw2) opsin transcripts, but lws opsin transcripts (lws1/opn1lw1, lws2/opn1lw2) were not detected, suggesting that the procedure likely separated double cone pairs. Through this method, pure, sorted cell samples can provide RNA that is reliable for downstream gene expression analyses, such as qRT-PCR and RNA-seq, which may reveal molecular signatures of photoreceptors and microglia for comparative transcriptomics studies.