RNA Isolation Techniques Research Articles

Modification of the Trizol Method for the Extraction of RNA from Prorocentrum triestinum ACIZ_LEM2.

In samples of harmful algal blooms (HABs), seawater can contain a high abundance of microorganisms and elemental ions. Along with the hardness of the walls of key HAB dinoflagellates such as Prorocentrum triestinum, this makes RNA extraction very difficult. These components interfere with RNA isolation, causing its degradation, in addition to the complex seawater properties of HABs that could hinder RNA isolation for effective RNA sequencing and transcriptome profiling. In this study, an RNA isolation technique was established through the modification of the Trizol method by applying the Micropestle System on cell pellets of P. triestinum frozen at -20 °C, obtained from 400 mL of culture with a total of 107 cells/mL. The results of the modified Trizol protocol generated quality RNA samples for transcriptomics sequencing, as determined by their measurement in Analyzer Agilent 4150.

Effective RNA isolation method for gram-positive and acid-fast bacteria: metamorphosed from conventional RNA isolation techniques.

The RNA-based study provides an excellent indication of an organism's gene expression profile. Obtaining high-yield and high-purity RNA from Gram-positive and acid-fast bacteria is difficult without high-end kits and facilities. We optimised effective and simple protocol for RNA isolation that is a combination of enzymatic, physical and chemical treatment to disrupt cells. We successfully isolated high quality intact total RNA with yields ranging from 23.13 ± 0.40 to 61.51 ± 0.27µg and the 260/280 purity ratio of 1.95 ± 0.01 to 2.05 ± 0.01 from Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Mycobacterium smegmatis. These results represents a significantly enhanced yield and purity compared to other combination of techniques which we performed. Compared to previous studies the yield obtained by this method is high for the studied organisms. Furthermore the yielded RNA was successfully used for downstream applications such as quantitative real time PCR. The described method can be easily optimised and used for various bacteria.

Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues.

Improving RNA isolation and cDNA synthesis techniques has emerged due to advancements in the knowledge of molecular basis of most diseases. This in turn increased the need of higher quantity and quality of the extracted genetic material to be used for a variety of diagnostic tests and experiments. The aim of the study was to compare three modified methods for RNA extraction from formalin-fixed paraffin embedded (FFPE) biopsied tissue and different cDNA synthesis strategies to facilitate study of gene expression. Compared RNA extraction methods were: lysis buffer, phenol-based extraction, and combination of both with concomitant use of silica-based spin columns. RNA quantity and purity were estimated spectrophotometrically. Different priming strategies for cDNA synthesis were applied: oligodT, combination of oligodT and random hexamer, and gene specific primer. Two-step RT-qPCR of ribosomal protein L37A on preamplified and non-preamplified cDNA templates was performed. The combination of lysis buffer with phenol based extraction gave higher RNA yield. By doing cDNA preamplification, the confidence of detection by qPCR was raised, and efficiency was improved. The preamplified template increased the sensitivity of analysis. Together, the combination of approaches improved substantially the reproducibility and validity of quantitative gene expression analyses from FFPE tissues.

Environmentally friendly method of RNA isolation

The diversity of organisms, tissues and cells is so great that, to date, no universal method for RNA extraction from these biological materials exist. The RNA isolation technique with a mix of guanidine thiocyanate, phenol, and chloroform is most widely used. Extraction and purification of RNA methods using selling guanidinium-phenol (TRIzol)-based and silica-based column kits have limitations on toxicity, or RNA isolation, particularly for plants, and scaling. The agents' toxicity is particularly relevant when employing for mass analysis in practice while gaining RNA preparations during the pandemics, epizootics, and epiphytotic. In modern diagnostics of infections at the molecular level, powerful RT-PCR technology is used, which amplifies the detection of RNA pathogens by hundreds of millions of times. We proposed obtaining RNA samples from viruses, bacteria, and plants for the reverse transcription reactions with a subsequent amplification of cDNAs by the polymerase chain reaction using potent and nontoxic chaotropic agent ammonium trichloroacetate. The method works in the analytical and preparative range and can be useful in the case of extraordinary circumstances during mass infections. Potentially this method can be adapted for obtaining RNA samples ready for the RT-isothermal PCR in the field.

944P - Evaluation of different diagnostic methods for identification of FGFR alteration in advanced urothelial carcinomas: Proficiency results based on multiple RNA extraction kits and mutation detection methods

BackgroundPan FGFR inhibitors such as Erdafitinib are approved in patients with advanced urothelial carcinoma (aUC) harboring FGFR alterations. Based on the necessity of sensitive and reproducible identification of these alterations in diagnostic routines this study aimed to compare different RNA isolation techniques and the QIAGEN therascreen® FGFR RGQ RT-PCR Kit with the SNaPshot mutational analysis. MethodsNucleic acids were extracted from 47 UC patients using the QIAGEN RNeasy® DSP FFPE Kit, STRATIFYER Xtract or Maxwell RNA (Promega) isolation kit. Mutations of the FGFR3 gene were detected using the SNaPshot method as well as via the QIAGEN therascreen® FGFR RGQ RT-PCR Kit. Intrinsic molecular subtypes were assessed using immunohistochemistry (IHC) of GATA3, FOXA1, CK20, CK5 and CD44 as well as RT-qPCR for gene expression of CK20 and CK5. ResultsAll three different RNA isolation kits showed comparable amount of extracted RNA (Median 290, 177 and 226ng/ul). Six hotspot FGFR3 mutations were identified with 100% concordance. The main difference were the necessary working hours: per 10 samples, 4 vs. 4.5 vs 8hours were needed for Stratifyer, Maxwell or QIAGEN RNA isolation. In addition, of 47 analyzed samples 100% concordance between QIAGEN therascreen® FGFR RGQ RT-PCR Kit and SNapShot analysis were achieved with 18 mutations identified (14 S249C, 2 R248C and 2 Y375). Immunohistochemical basal marker profile (CD44 and CK5) as well as high CK5 expression was observed in 28 (6/28 FGFR3-altered). Luminal marker profile (IHC markers: GATA3, FOXA1 and CK20 as well as expression of CK20) was detected in 37 samples (5/37 with FGFR3 alteration, n.s.). ConclusionsDifferent RNA extraction methods presented with comparable amount of nucleic acids with variant working hours. Moreover, a 100% concordance between the well established SNaPshot analysis compared to the QIAGEN therascreen® FGFR RGQ RT-PCR Kit for FGFR3 mutations was observed. So far, the frequency of FGFR3 mutations in aUC was not related to the type of UC as defined by marker profils. Legal entity responsible for the studyBRIDGE consortium. FundingJanssen. DisclosureR. Wirtz: Officer / Board of Directors: Stratifyer. M. Eckstein: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Janssen. A. Hartmann: Advisory / Consultancy: Janssen. All other authors have declared no conflicts of interest.

Interleukin-17A affects extracellular vesicles release and cargo in human keratinocytes.

Psoriasis is a chronic inflammatory systemic disease caused by deregulation of the interleukin-23/-17 axis that allows the activation of Th17 lymphocytes and the reprogramming of keratinocytes proliferative response, thereby inducing the secretion of cyto-/chemokines and antimicrobial peptides. Beside cell-to-cell contacts and release of cytokines, hormones and second messengers, cells communicate each other through the release of extracellular vesicles containing DNA, RNA, microRNAs and proteins. It has been reported the alteration of extracellular vesicles trafficking in several diseases, but there is scarce evidence of the involvement of extracellular vesicles trafficking in the pathogenesis of psoriasis. The main goal of the study was to characterize the release, the cargo content and the capacity to transfer bioactive molecules of extracellular vesicles produced by keratinocytes following recombinant IL-17A treatment if compared to untreated keratinocytes. A combined approach of standard ultracentrifugation, RNA isolation and real-time RT-PCR techniques was used to characterize extracellular vesicles cargo. Flow cytometry was used to quantitatively and qualitatively analyse extracellular vesicles and to evaluate cell-to-cell extracellular vesicles transfer. We report that the treatment of human keratinocytes with IL-17A significantly modifies the extracellular vesicles cargo and release. Vesicles from IL-17A-treated cells display a specific pattern of mRNA which is undid by IL-17A neutralization. Extracellular vesicles are taken up by acceptor cells irrespective of their content but only those derived from IL-17A-treated cells enable recipient cells to express psoriasis-associated mRNA. The results imply a role of extracellular vesicles in amplifying the pro-inflammatory cascade induced in keratinocyte by pro-psoriatic cytokines.

An Efficient Approach for RNA Extraction from Boar Sperm and Seminal Plasma.

Despite transcriptional silencing in mature sperm and cytoplasmic expulsion of RNA during the final sperm maturation process, thousands of RNAs have been successfully identified in ejaculated sperm. Although most of RNAs' function is still unknown, it is suggested that sperm RNAs have a vital biological role in fertilization and post-fertilization events. Nevertheless, the lack of accurate RNA isolation techniques and the resultant good quality sperm RNA has hampered the exploration of sperm RNAs function. Additionally, small non-coding RNAs are found in extracellular fluids including seminal plasma. These small RNAs may participate in cell to cell communication or intracellular and extracellular message transmission. Developing precise protocols to extract RNA from sperm and seminal plasma is critical to elucidate sperm physiology and paternal contributions to fertilization and post-fertilization events. A detailed procedure consisting of semen collection, separation of sperm and seminal plasma, extracting RNA from sperm and seminal plasma, and determining the quantity and quality of RNA for boar semen is presented here. This efficient protocol can be extrapolated to isolate RNAs from sperm and seminal plasma across mammalian species.

High-quality human and rat spermatozoal RNA isolation for functional genomic studies.

Sperm RNA is a sensitive monitoring endpoint for male reproductive toxicants, and a potential biomarker to assess male infertility and sperm quality. However, isolation of sperm RNA is a challenging procedure due to the heterogeneous population of cells present in the ejaculate, the low yield of RNA per spermatozoon, and the absence of 18S and 28S ribosomal RNA subunits. The unique biology of spermatozoa has created some uncertainty in the field about RNA isolation methods, indicating the need for rigorous quality control checks to ensure reproducibility of data generated from sperm RNA. Therefore, we developed a reliable and effective protocol for RNA isolation from rat and human spermatozoa that delivers highly purified and intact RNA, verified using RNA-specific electrophoretic chips and molecular biology approaches such as RT-PCR and Western blot analysis. The sperm RNA isolation technique was optimized using rat spermatozoa and then adapted to human spermatozoa. Three steps in the sperm isolation procedure, epididymal fluid collection, sperm purification, and spermatozoon RNA extraction, were evaluated and assessed. The sperm RNA extraction methodology consists of collection of rat epididymal fluid with repeated needle punctures of the epididymis, somatic cell elimination using detergent-based somatic cell lysis buffer (SCLB) and the use of RNA isolation Kit. Rat sperm heads are more resistant to disruption than human spermatozoa, necessitating the addition of mechanical lysis with microbeads and heat in the rat protocol, whereas the human sperm protocol only required lysis buffer. In conclusion, this methodology results in reliable and consistent isolation of high-quality sperm RNA. Using this technique will aid in translation of data collected from animal models, and reproducibility of clinical assessment of male factor fertility using RNA molecular biomarkers.

Don't just dump your data and run: Authors should submit as much experimental information as possible when uploading sequence data.

If you have, in any way, been involved with genetic research over the past 10 years, then you have likely heard of the Sequence Read Archive (SRA), which is jointly housed at the National Center for Biotechnology (NCBI), the DNA Data Bank of Japan (DDBJ), and the European Bioinformatics Institute (EBI). And if you regularly work with genome or transcriptome sequence information, then you have probably extracted data from and/or deposited data into the SRA. For those who are unfamiliar with it, the SRA is an international public online archive for next‐generation sequencing (NGS) data, which was established about a decade ago under the guidance of the International Nucleotide Sequence Database Collaboration (INSDC) [1], [2]. Despite nearly being shut down in 2011 [3], it has grown at a staggering rate over the past 10 years. As of September 1, 2017, it housed over five quadrillion (1015) open‐access bases of NGS data, coming from thousands of different species and spanning the entire gamut of cellular and viral life. It contains DNA‐ and RNA‐sequencing (DNA‐seq and RNA‐seq) reads of every kind, from bisulfite‐seq to strand‐specific RNA‐seq to single‐cell DNA‐seq, and it accepts reads from every type of NGS platform, be it Illumina, Ion Torrent, or PacBio sequencing. In other words, the SRA is a crucial and central resource in the fast‐paced and increasingly important domain of contemporary genetic research. > … the SRA is a crucial and central resource in the fast‐paced and increasingly important domain of contemporary genetic research. ### The Sequence Read Archive The SRA can be easily accessed and searched via the NCBI (https://www.ncbi.nlm.nih.gov/sra), DDBJ (http://trace.ddbj.nig.ac.jp/dra/index_e.html), and EBI (http://www.ebi.ac.uk/ena/submit/read-submission) websites. Once there, you will find yourself at a sequencing‐read superstore. With a decent Wi‐Fi connection, a couple of keyword searches, and a few clicks of the trackpad, you can quickly …

RNA isolation from paraffinated material of histological´s prepared slides

The parafinization of solid biopsies allows a better preservation and preparation of microscopy slides used for histological evaluations. Techniques of RNA extraction are suitable to be applied in microtomized paraffinated material, but the extraction of paraffinic material already disposed in a slide has not yet been described and can be useful for gene expression studies. We adapted the RNA isolation technique of paraffinated material for slide-laid paraffinited material, using the Rneasy® FFPE Kit. Total RNA so isolated is suitable to gene expression analysis by RT-qPCR.

A disposable lab-on-a-chip platform for highly efficient RNA isolation

Despite recent advances in lab-on-a-chip (LOC) technology, RNA-based miniaturized diagnostic platforms have been falling behind due to a lack of on-chip RNA isolation techniques. However, RNA analysis plays an important role in gene expression determination and pathogen detection. To meet the increasing need for the analysis of RNA with point-of-care (POC) testing, a reliable LOC platform for RNA isolation needs to be developed. Here, we present a method of RNA isolation using a LOC platform that provides high-quality RNA from a variety of biological samples. By taking advantage of pH-dependent reversible dimethyl adipimidate (DMA) binding to RNA, we were able to perform RNA isolation on a disposable LOC platform within 30min without the need for chaotropic salts or solvent. Using this LOC platform, gene expression analyses were successfully carried out with RNA isolated from different cell lines (HEK293, Caco-2, MCF-7, and T24) by subsequent real-time reverse transcription PCR. In addition, we validated the utility of this LOC platform with viral RNA isolated from Dengue viruses (DENV-1, −2, −4). We demonstrated a high recovery rate and rapid processing with minimal contamination. Therefore, this novel method could be an ideal RNA isolation technique in gene expression research and for the diagnosis of infectious diseases caused by RNA viruses in POC testing.

Isolation of Mature Adipocytes from White Adipose Tissue and Gene Expression Studies by Real-Time Quantitative RT-PCR.

The study of adipose tissue and more specifically of adipocytes is considered pivotal for dissecting molecular mechanisms responsible for alterations in several organs and systems, including adipose tissue, not only in obesity but also in other diseases (hypertension, heart failure). Adipose tissue is a complex tissue composed of adipocytes and the stromal vascular fraction which includes a heterogeneous population of preadipocytes, blood cells, endothelial cells, and macrophages. In the present chapter, methods are detailed to generate purified mature adipocytes from white adipose tissue by using enzymatic digestion. Such methods should help laboratories to study the specific roles of adipocytes in different pathologies and are easily adaptable to different animal models. Moreover, as gene activity is controlled at both transcriptional and posttranscriptional levels, it is very important to determine the levels of messenger ribonucleic acid (mRNA) of genes of interest. This process involves the isolation of total RNA and subsequent analysis of the mRNA of interest by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Given the unique nature of adipose tissue and adipocytes (i.e., containing high amounts of lipid), we have set up a special RNA isolation technique in both white adipose tissue and isolated mature adipocytes from mice. In summary, isolation and culture of adipocytes in vivo and gene expression studies will help to understand the mechanisms that control adipocyte function in physiological and pathological states and may lead to design interventions that might affect the adipocyte birth-death balance or phenotype.

Plattformunabhängige Isolierung hochmolekularer Nukleinsäuren

More than 35 years after describing the first silica-based DNA and RNA isolation technique, novel methods in the field of nucleic acid extraction are invented now. The new method is especially in keeping with the trend toward automating processes and fullfil the need for qualitative, high molecular weight DNA, which gain importance in Next Generation Sequencing (NGS).

Abstract 5247: Comprehensive and systematic identification of miR-215 targets in colon cancer via a novel translation capture immunoprecipitation (TrIP) approach

Abstract The important roles that non-coding miRNAs play in cancer development, makes the investigation of the various genes they target particularly crucial. In addition to targeting mRNA transcripts for degradation, miRNA can also alter gene expression at the translational level to achieve rapid regulation. It has been a technical challenge to study translational control using a small number of cells. We have overcome this hurdle by developing a translational capture immunoprecipitation approach (TrIP) to investigate RNA binding proteins or non-coding RNA regulation at the translational level from a small numbers of cells. The new approach is based on the fact that nascent translated polypeptides are associated with hsp70 chaperones along with actively translating mRNA transcript complexes, called polyribosomes. We are able to isolate actively translating mRNA from free cytoplasmic mRNA by immunoprecipitation of hsp70 associated polyribosome complexes. In this study, we utilize this novel approach to identify both degraded and non-degraded mRNA transcripts targeted by miR-215 in colon cancer. Following miR-215 overexpression in colon cancer cells, actively translating mRNA transcripts were isolated using the TrIP technique and purified following standard RNA isolation techniques. We were able to identify additional non-degraded mRNA targets regulated by miR-215 using a TrIP with transcriptome-wide microarray based approach, when compared to a generic approach based on steady state total RNA that can only identify degraded targets. Specific targets were independently validated using qRT-PCR analysis. We further validated miR-215 targets at the protein level with a comprehensive and high-throughput quantitative SILAC proteomics analysis. Protein targets were further validated using Western blotting with select positive control targets including TYMS, DTL and DHFR. This approach offers the unique opportunity to understand not only the mRNA targets a miRNA may target for degradation, but also those it regulates at the translational level, providing unique insight into the actions of miRNAs. Interest in this type of thorough analysis of different miRNAs or RNA binding proteins will likely continue to grow as the important roles miRNA play in cancer are further investigated. Citation Format: Andrew Fesler, Xiao Xu, Emily Chen, Jingfang Ju. Comprehensive and systematic identification of miR-215 targets in colon cancer via a novel translation capture immunoprecipitation (TrIP) approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5247. doi:10.1158/1538-7445.AM2014-5247

Abstract 3506: mRNA biomarkers of colorectal cancer development tested on biopsy, fresh frozen and formalin-fixed, paraffin embedded (FFPE) specimens in an automated workflow.

Abstract Introduction: The early molecular detection of the colorectal dysplasia-carcinoma transition may enhance the classification of colonic tissue samples. Recently, a colorectal cancer-specific transcript set including COL12A1, CXCL2, CA7, Il1B, MMP3, IL8 was identified on colonic biopsy samples using whole genomic microarrays (WG MA). Aims: Our aim was to evaluate the applicability of these markers on independent biopsy and also on fresh frozen and formalin-fixed, paraffin-embedded (FFPE) tissue specimens from healthy and CRC patients by expression arrays and RT-PCR. Furthermore, automated RNA isolation was introduced and evaluated for increased sample number in one run. Materials and methods: Total RNA from 3 normal and 3 CRC FFPE specimens were analyzed by using Affymetrix U133Plus 2.0 WG expression arrays. Total RNA was isolated from 30 biopsy samples stored in RNALater (15 CRC, 15 normal), 20 fresh frozen surgically removed colonic tissues (10 CRC, 10 adjacent normal) and 60 FFPE (30 CRC, 30 adjacent normal) samples with the automated MagNA Pure 96 Cellular RNA Large volume kit and reverse transcription was done using Transcriptor First Strand cDNA Synthesis Kit (Roche). Gene expression analysis was performed with real-time PCR using RealTime ready assays and the LightCycler 480 system (Roche). Results: The FFPE specimens could be clustered correctly in 100% by the results of the microarray analysis. According to the gene expression levels of the set of 11 transcripts, the biopsy samples could be distinguished by 93,3% sensitivity and 93,3% specificity by RT-PCR. The discriminatory power of the marker set was proved to be high also on fresh frozen and FFPE samples (sens:>90 %, spec: >80 %). The automated RNA isolation technique has not influenced the classification power of the markers, but decreased the workaround time by 100% (4 hours to 2 hours). Conclusion: The analyzed set of markers in an automated environment could correctly characterize the healthy and the tumorous colonic tissue samples on biopsy, fresh frozen and also on FFPE tissue samples by MAs and RT-PCR. On the basis of these results, these markers can enhance the automated, early and differential detection of colorectal cancer. Citation Format: Bela Molnar, Alexandra Kalmar, Barnabás Wichmann, Orsolya Galamb, Sandor Spisak, Ferenc Sipos, Kinga Tóth, Katalin Leiszter, Arpad V. Patai, Andrea Schöller, Zsolt Tulassay. mRNA biomarkers of colorectal cancer development tested on biopsy, fresh frozen and formalin-fixed, paraffin embedded (FFPE) specimens in an automated workflow. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3506. doi:10.1158/1538-7445.AM2013-3506

Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels

Extracting high-quality RNA from hydrogels containing polysaccharide components is challenging, as traditional RNA isolation techniques designed for cells and tissues can have limited yields and purity due to physiochemical interactions between the nucleic acids and the biomaterials. In this study, a comparative analysis of several different RNA isolation methods was performed on human adipose-derived stem cells photo-encapsulated within methacrylated glycol chitosan hydrogels. The results demonstrated that RNA isolation methods with cetyl trimethylammonium bromide (CTAB) buffer followed by purification with an RNeasy® mini kit resulted in low yields of RNA, except when the samples were preminced directly within the buffer. In addition, genomic DNA contamination during reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was observed in the hydrogels processed with the CTAB-based methods. Isolation methods using TRIzol® in combination with one of a Qiaex® gel extraction kit, an RNeasy® mini kit, or an extended solvent purification method extracted RNA suitable for gene amplification, with no evidence of genomic contamination. The latter two methods yielded the best results in terms of yield and amplification efficiency. Predigestion of the scaffolds with lysozyme was investigated as a possible means of enhancing RNA extraction from the polysaccharide gels, with no improvements observed in terms of the purity, yield, or amplification efficiency. Overall, this work highlights the application of a TRIzol®+extended solvent purification method for optimizing RNA extraction that can be applied to obtain reliable and accurate gene expression data in studies investigating cells seeded in chitosan-based scaffolds.

Detection of Low‐concentration Host mRNA Transcripts in Malawian Children at Risk for Environmental Enteropathy

Transcriptomic analysis of fecal samples is an emerging method for the diagnosis of gastrointestinal pathology because it is noninvasive and requires minute volumes of analyte; however, detection of mRNA in low copy numbers in human stool is challenging. Our objective was to develop a method for detecting human mRNA suggestive of environmental enteropathy (EE) in feces. Stool samples from 70 Malawian children, 34 without EE and 36 with EE, as defined by dual sugar absorption, were used to develop the methodology for mRNA detection. Multiple RNA isolation techniques and polymerase chain reaction formats were tested to detect 38 potential mRNA biomarkers suggestive of EE, and the results compared. RNA isolation using magnetic bead extraction best recovered host mRNA in stool, and digital droplet polymerase chain reaction was the most sensitive format to detect low copy numbers of mRNA. In all of the 70 samples, >20 copies of glyceraldehyde-3-phosphate dehydrogenase/200 mg of stool were detected. Copy numbers of potential biomarkers were normalized to glyceraldehyde-3-phosphate dehydrogenase, to account for interspecimen differences in concentration of human mRNA. Of the 38 transcripts chosen for initial evaluation, 24 had copy numbers >10 in all of the samples tested. Of the 6 potential markers measured in all of the 70 samples, REG4 best differentiated children with and without EE. A reproducible and reliable method to quantify human mRNA in stool present in low copy numbers has been developed, and may prove useful in investigations of EE and possibly other inflammatory gut conditions.

Laser Capture Microdissection of <em>Drosophila</em> Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser capture microdissection of Drosophila peripheral neurons.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification, Fluorescent Activated Cell Sorting (FACS), and RNA binding protein based strategies, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells, including da neurons at the third instar larval stage of development. Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP transgene under the control of either the class IV da neuron specific ppk-GAL4 driver or the pan-da neuron specific 21-7-GAL4 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

The untranslated side of hair and skin mammalian pigmentation: Beyond coding sequences

For several decades, tremendous advances in studying skin and hair pigmentation of mammals have been made using Mendelian genetics principles. A number of loci and their associated traits have been extensively examined, crossings performed, and phenotypes well documented. Continuously improving PCR techniques allowed the molecular cloning and sequencing of the first pigmentation genes at the end of the 20th century, a period followed by an intense effort to detect and describe polymorphisms in the coding regions and correlate allelic combinations with the observed melanogenic phenotypes. However, a number of phenotypes and biological events could not be elucidated solely by analysis of the coding regions of genes. Messenger RNA isolation, characterization and quantification techniques allowed groups to move ahead and investigate molecular mechanisms whose secrets lay within the noncoding regions of pigmentation genes transcripts such as MC1R, ASIP, or Mitf. The untranslated elements contain specific nucleotidic sequences and structures that dramatically influence the mRNA half-life and processing thus impacting protein translation and melanin production. As we are progressively uncovering the complex processes regulating melanocyte biology, unraveling complete mRNA structures and understanding mechanisms beyond coding regions has become critical.