Small RNA Samples Research Articles

MiRNA Predictors of Response to DNA Methyltransferase Inhibitors

BackgroundMyelodysplastic syndrome (MDS) is a common disease of the elderly characterized by ineffective maturation of hematopoietic cells manifesting as low blood counts, morphologic atypia, and predisposition to the development of acute leukemia. The only FDA-approved drugs for the treatment of all subytpes of MDS are the DNA methyltransferase inhibitors (DNMTis), azacytidine and decitabine. However, only a minority of patients achieve a hematologic response or better on these agents. We have previously identified a miRNA signature which distinguishes MDS patients from normal controls. We sought to investigate whether miRNA expression might also be predictive of how patients respond to therapy with DNA methyltransferase inhibitors (DNMTis).DesignWe collected paired fresh frozen bone marrow mononuclear cells (BM-MNCs) from 28 patients with MDS both pre and post treatment with DNMTis as well as from 30 normal controls. Following enrichment for miRNAs and other small RNA species using the TruSeq Small RNA sample preparation kit (Illumina, San Diego, CA), miRNAs were sequenced on an Illumina HiSeq 2500. Alignment was performed using Bowtie 2 against reference sequences from mirBASE and differential expression analysis was analyzed using a rank sum score from the following analysis programs: DESeq, edgeR and baySeq. The response to the DNMTis was characterized based upon the following scale: 1 = complete remission, 2 = marrow complete remission, 3 = partial remission, 4 = hematologic improvement, 5 = stable disease, and 6 = progressive disease. Linear regression analysis was conducted using the response score as outcome and miRNA expression value as the predictor.ResultsSeveral differences were found between the control samples and the pre-treatment MDS samples, many of which have been described previously, including hsa-miR-125b, hsa-miR-342, hsa-miR-140, and hsa-miR-150. Not unexpectedly, the expression of numerous miRNAs was significantly altered as a result of treatment with DNMTis. However, there were no significant miRNA expression differences between responders and non-responders in the post-treatment samples and no significant differences between the changes in miRNA expression (pre versus post treatment changes) in responders and non-responders. Interestingly, 5 miRNAs demonstrated significant association between their pre-treatment expression and the degree of response (FDR < 0.05): hsa-miR-125a, hsa-miR-342, hsa-miR-146b, hsa-miR-146a, hsa-miR-423, and hsa-miR-150. These miRNAs share striking overlap in their mRNA targets including key pathway regulators in the p53 pathway as well as numerous growth factors implicated in hematopoietic stem cells as well as hematopoietic stem cell markers.ConclusionThese studies confirm that miRNA expression differences differentiate between MDS and normal controls. The administration of DNMTis results in widespread expression changes regardless of the response to therapy. However, the expression of a small handful of miRNA are positively correlated with the degree of response in MDS pre-treatment samples to DNMTis and therefore may be useful as markers which predict responders to DNMTi therapy in addition to suggesting biological pathways implicated in the mechanism of response. These results await further prospective studies for confirmation. DisclosuresNo relevant conflicts of interest to declare.

MiRNA Genes of an Invasive Vector Mosquito, Aedes albopictus

Aedes albopictus, a vector of Dengue and Chikungunya viruses, is a robust invasive species in both tropical and temperate environments. MicroRNAs (miRNAs) regulate gene expression and biological processes including embryonic development, innate immunity and infection. While a number of miRNAs have been discovered in some mosquitoes, no comprehensive effort has been made to characterize them from different developmental stages from a single species. Systematic analysis of miRNAs in Ae. albopictus will improve our understanding of its basic biology and inform novel strategies to prevent virus transmission. Between 10–14 million Illumina sequencing reads per sample were obtained from embryos, larvae, pupae, adult males, sugar-fed and blood-fed adult females. A total of 119 miRNA genes represented by 215 miRNA or miRNA star (miRNA*) sequences were identified, 15 of which are novel. Eleven, two, and two of the newly-discovered miRNA genes appear specific to Aedes, Culicinae, and Culicidae, respectively. A number of miRNAs accumulate predominantly in one or two developmental stages and the large number that showed differences in abundance following a blood meal likely are important in blood-induced mosquito biology. Gene Ontology (GO) analysis of the targets of all Ae. albopictus miRNAs provides a useful starting point for the study of their functions in mosquitoes. This study is the first systematic analysis of miRNAs based on deep-sequencing of small RNA samples of all developmental stages of a mosquito species. A number of miRNAs are related to specific physiological states, most notably, pre- and post-blood feeding. The distribution of lineage-specific miRNAs is consistent with mosquito phylogeny and the presence of a number of Aedes-specific miRNAs likely reflects the divergence between the Aedes and Culex genera.

One-step real time RT-PCR for detection of microRNAs

Rapid and simple methods for microRNA (miRNA) detection are essential for biological research of miRNAs and clinical diagnosis. Here we describe a sensitive and specific real time RT-PCR (also RT-qPCR) method for miRNA quantification. The whole detection process including reverse transcription and PCR is performed in one PCR tube by a one-step operation on a real-time PCR system. The results display a wide linear range from 0.1amol to 10fmol with a detection limit of 12.6zmol for miRNA let-7a detection. Let-7a in small RNA samples extracted from tumor cells has been successfully detected by this method. This method is cost-effective, simple and rapid, and has the advantages in the high-throughput routing assay of given miRNAs, as well as in non-model research that has less specific kits and reagents.

Purification and Concentration of Nucleic Acids

AbstractThe purification and concentration of nucleic acids have become routine procedures in most biology and molecular biology laboratories. This unit covers the basic principles and procedures for the isolation, purification, and manipulation of solutions of DNA or RNA. The basic DNA protocol, using phenol extraction and ethanol precipitation, is appropriate for the purification of DNA from small volumes (&lt;0.4 ml) at concentrations ≤1 mg/ml. Purification of DNA using commercially available silica membrane spin columns is presented as an alternate protocol. Isolation and purification of RNA from mammalian cells or tissues is also examined. Use of the protein denaturant guanidine thiocyanate and water‐saturated phenol, followed by concentration by isopropanol precipitation, for producing small samples of RNA, is illustrated in the basic RNA protocol. Curr. Protoc. Essential Lab. Tech. 6:5.2.1‐5.2.21. © 2012 by John Wiley &amp; Sons, Inc.

Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome

RNA editing is a post-transcriptional event that recodes hereditary information. Here we describe a comprehensive profile of the RNA editome of a male Han Chinese individual based on analysis of ∼767 million sequencing reads from poly(A)(+), poly(A)(-) and small RNA samples. We developed a computational pipeline that carefully controls for false positives while calling RNA editing events from genome and whole-transcriptome data of the same individual. We identified 22,688 RNA editing events in noncoding genes and introns, untranslated regions and coding sequences of protein-coding genes. Most changes (∼93%) converted A to I(G), consistent with known editing mechanisms based on adenosine deaminase acting on RNA (ADAR). We also found evidence of other types of nucleotide changes; however, these were validated at lower rates. We found 44 editing sites in microRNAs (miRNAs), suggesting a potential link between RNA editing and miRNA-mediated regulation. Our approach facilitates large-scale studies to profile and compare editomes across a wide range of samples.

Abstract B36: Micronome-wide explorations of NSCLC: microRNA-seq discovery

Abstract Background: Recent studies have revealed that microRNAs play important regulatory roles in carcinogenesis. Some key miRNAs may regulate lung carcinogenesis and be useful for lung cancer early detection. For discovery, we used high-throughput next-gen sequencing technology to investigate miRNA expression profiles in a study of 25 lung adeno- and squamous cell carcinomas, paired with far-adjacent noncancerous lung tissues from the same donors. Method: Total RNA was isolated by Trizol from 14 pairs of lung adenocarcinomas, 11 squamous cell cancers, and paired noncancerous lung tissues, from the same patients. Small RNA library of each total RNA was constructed with Illumina's Small RNA Sample Prep Kit, followed by high-throughput sequencing using the Illumina IIa platform. In parallel, transcriptome microarrays (Affymetrix GA 1.0) were run on these same paired samples. Statistical differences in microRNA expression between groups were analyzed by GenePattern software, logistic regression, and other methods. Results: For adeno, 128 micros were differentially expressed and 63 were two-fold differentially expressed between the tumors and non-tumor tissue; for squamous, the numbers of altered microRNAs were 42 overall, and 25 &amp;gt;two-fold. Among adenocarcinoma, the top 10 upregulated were miRs-147b, 577, 877, 556, 449c, and others; and downregulated were miRs 516b, 1251, 486, 139, 516a, and others. Among squamous cell carcinomas, the top up-regulated miRs were 708, 1259, 494, 200a, 216b, and others; and down-regulated miRs were 184, 1251, 144, 516a, 584, and others. MiRs 1259, 135b, 200a, 21, 494, 708 were upregulated in both tumor histologies. When the highest expressed microRNAs were correlated with the most altered mRNA transcripts, putative mRNA targets were more often anti-correlated to the individual microRNAs (Chi Square, p = 4.6 × 10-6), and this anticorrelation generally held true for the top differentially expressed microRNAs and their putative mRNA targets, and within histology strata. Conclusion: The lung cancer microRNA complement differs from non-tumor tissue, adeno- differs from squamous, and many of the most dysregulated miRs appear to be anticorrelated with putative mRNA targets. Which of the putative mRNA targets are actual biological targets awaits experimental functional work. Once constructed, the combined microRNA:mRNA signature of lung cancer is likely to be more informative for biologic potential than either signature alone. [Funding source: NCI 1RC1 CA145422-01; 1K24-CA139054-01]

Using deep sequencing of formalin-fixed paraffin embedded (FFPE) rectal cancer biopsy tissue to analyze miRNA expression in patients with rectal cancer.

487 Background: miRNAs have been shown to be involved in tumor initiation, progression and metastasis in many cancers including colorectal cancer. However, miRNA profiling specifically in rectal cancer is not well characterized. Our objective was to generate a miRNA expression profile in locally advanced rectal cancer using formalin fixed paraffin embedded (FFPE) biopsy tissue collected from patients with locally advanced rectal cancer. Methods: We collected pre-treatment biopsy tissue and matched normal tissue from 40 rectal cancer patients treated with pre-operative chemoradiation (CRT) and total mesorectal excision (TME). We extracted 50-1000ng of total RNA from FFPE biopsies and optimized small RNA sample preparation for deep-sequencing. We then performed deep sequencing on biopsy and matched normal tissue and compared miRNA expression in biopsy and normal tissues using paired T-test and multiple testing (Q-bound &lt;0.05) to determine significant miRNA expression changes in rectal cancer. Results: 182 miRNAs were differentially expressed in tumor versus normal tissues (Q-bound &lt;0.05); 15 of these miRNAs showed a greater than 2-fold change in expression in tumor tissue; mir-18a, mir-135b, mir-503, mir-584, mir-106b, mir-224, mir-92a, mir-181d were up-regulated and mir-375, mir-378, mir-378c, mir-137, mir-378, mir-147b, mir-30a were down-regulated. miRNA mir-31 showed the highest up-regulation in tumor tissue (16-fold increase) while mir-215 expression decreased 8-fold in tumor compared to normal tissue. Tumor and normal tissues were completely separated by hierarchical cluster analysis based on their distinctive miRNA profiles. Conclusions: We optimized small RNA sample preparation for deep sequencing of miRNAs, an approach which may be useful for quantifying miRNA expression in tissues with limited starting material. We also identified a novel miRNA expression profile in rectal cancer that may be useful as a diagnostic biomarker of disease.

Regulatory network in lignin biosynthetic pathway through small RNAs in Acacia mangium: implications to the pulp and paper industry

Background Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component of wood, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is a costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the monolignol biosynthesis pathway genes [1-3]. Non-coding RNAs represent an emerging class of riboregulators, which are processed to shorter miRNAs or siRNAs. The current paradigm indicated that plant system use small RNAs (miRNAs and siRNAs) as guide for post-transcriptional gene silencing and epigenetic regulation. Although miRNAs and siRNAs result from different biogenesis pathways but both interact with target transcripts for direct cleavage or translation repression, effectively shutting down that genes’ functions. However, much less is known about the mechanism of gene regulation governed by these small RNAs in lignin biosynthesis pathway in A. mangium. Methods Total RNA was isolated from secondary xylem tissue with contrasting lignin content using mirVana microRNA Isolation Kit (Cat. AM1561, Ambion, Austin, TX, USA) following manufacturer’s protocol. Thin cookies were first ground in a blender and then further ground to fine powder using mortar and pestle. Integrity of the isolated Total RNA was analyzed using Bioanalyser 2100 (Agilent Technology, Palo Alto, USA) and only Total RNA with RIN value above 7 was selected for library construction. Library construction, sequencing and bioinformatics pipeline analysis was done by Gene Pool Sequencing Centre, United Kingdom. Small RNA libraries were generated using DGE small RNA Sample Preparation Kit (Cat. # FC-102-1009; Illumina, San Diego, CA, USA). Illumina sequencing libraries were prepared using the ‘long’ IIIumina protocol according to the manufacturer’s direction and two libraries were sequenced on an IIIumina GA-II following manufacturer’s instructions. After masking of adapter sequences and removal of contaminated reads, the clean reads were filtered and the resulting oligos were totalled. Clustering based on relative lengths from 7 nt to 35 nt were done using in house perl scripts. The targets were extracted by in house python script and annotated using BLAST to GO database. Only highly conserved miRNAs with strong differences in their expression level between high and low lignin secondary xylem were selected for validation using IQ5 real time – PCR technology (BioRad, Hercules, USA) (Table 1).

A simple and efficient method for isolating small RNAs from different plant species

BackgroundSmall RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.ResultsWe developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzol® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays.ConclusionHere we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays.

Glucocorticoids Modulate MicroRNA Expression and Processing during Lymphocyte Apoptosis

Glucocorticoids modulate immune development and function through the induction of lymphocyte apoptosis via mechanisms requiring alterations in gene expression. Recently, short, noncoding, microRNAs have been identified as key regulators of lymphocyte function; however, it is unknown whether glucocorticoids regulate noncoding microRNAs and whether this regulation contributes to lymphocyte apoptosis. We now show by both microarray and deep sequencing analysis that microRNAs are substantially repressed during glucocorticoid-induced apoptosis of primary rat thymocytes. Mechanistic studies revealed that primary microRNA transcripts were not repressed, whereas the expression of the key microRNA processing enzymes: Dicer, Drosha, and DGCR8/Pasha, were significantly reduced at both the mRNA and protein levels during glucocorticoid-induced apoptosis. To delineate the role of Dicer depletion and microRNA repression in apoptosis, we silenced Dicer expression in two human leukemic cell lines and demonstrated that Dicer depletion significantly enhanced glucocorticoid-induced apoptosis in both model systems. Finally, in vitro and in vivo overexpression of the conserved miR-17-92 polycistron, which was repressed significantly by dexamethasone treatment in both our microarray and deep sequencing studies, blunted glucocorticoid-induced apoptosis. These studies provide evidence of altered post-transcriptional microRNA expression and the repression of the microRNA bioprocessing pathway during glucocorticoid-induced apoptosis of lymphocytes, suggesting a role for microRNA processors and specific microRNAs in cell life/death decisions.

Identification of 21 microRNAs in maize and their differential expression under drought stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. The negative regulation of miRNAs has been found in response to stress in Arabidopsis, rice and other plants. The sequence of the maize genome has been completed very recently; however, only a few maize miRNAs have been identified and deposited in the miRNA registry database, and the details of their regulation of target genes remain to be elucidated. We identified 21 maize miRNAs using bioinformatics computation and small RNA cloning. They are designated as zma-miR161, zma-miR397, zma-miR446, zma-miR479, zma-miR530, zma-miR776, zma-miR782, zma-miR815a, zma-miR818a, zma-miR820, zma-miR828, zma-miR834, zma-miR1, zma-miR2, zma-miR3, zma-miR4, zma-miR5, zma-miR6, zma-miR7, zma-miR8 and zma-miR9. The characterization of these miRNAs reveals a length of 19–24 nt and the typical secondary structure. Using reverse transcriptional polymerase chain reaction (RT-PCR), 13 of these miRNAs were amplified and validated from total small RNA samples of maize. zma-miR1, zma-miR3, zma-miR6, zma-miR479, zma-miR782, zma-miR815a and zma-miR820 were found to be expressed specifically in drought-tolerant inbred line 87-1 under simulated drought stress, whereas the other six were amplified in line 87-1 and in the sensitive inbred line 200B under drought stress and in a well-watered control. The seven differentially expressed miRNAs are suggested to be associated with the regulation of gene expression under drought stress. Using the plant miRNA target finder software miRU and miRU2, 14 of the 21 miRNAs were identified to target genes encoding 21 proteins, most of which are metabolic enzymes. Seven of them are involved in signal transduction, transcription regulation, and bio- or abio-stress responses. The identification and characterization of 21 miRNAs enrich the maize miRNA database and provide valuable information for functional studies of miRNAs. Key words: MicroRNA, maize, bioinformatics, gene cloning, differential expression, drought stress.

Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples

BackgroundThe template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatamerization of the TS oligo.ResultsIn this study, we describe the application of nucleotide isomers that form non-standard base pairs in the template switching oligo to prevent background cDNA synthesis. When such bases are added to the 5' end of the template switching (TS) oligo, they inhibit MMLV-RT from extending the cDNA beyond the TS oligo, thus increasing cDNA yield by reducing formation of concatamers of the TS oligo that are the source of significant background.ConclusionsOur results demonstrate that this novel approach for cDNA synthesis has valuable utility for application of ultra-high throughput technologies, such as whole transcriptome sequencing using 454 technology, to very small biological samples comprised of tens of cells as might be obtained via approaches like laser microdissection.

Direct, Electronic MicroRNA Detection for the Rapid Determination of Differential Expression Profiles

In the time it takes for lunch: An electronic chip featuring nanostructured microelectrodes (NMEs) enables the analysis of microRNA expression profiles in just 30 min in small RNA samples without enzymatic amplification or sequence labeling. The multiplexed chip detects the hybridization of microRNA targets to NME surfaces and provides large electrocatalytic gain through the use of an ultrasensitive redox reporter system (see picture).

Direct RNA sequencing

Our understanding of human biology and disease is ultimately dependent on a complete understanding of the genome and its functions. The recent application of microarray and sequencing technologies to transcriptomics has changed the simplistic view of transcriptomes to a more complicated view of genome-wide transcription where a large fraction of transcripts emanates from unannotated parts of genomes, and underlined our limited knowledge of the dynamic state of transcription. Most of this broad body of knowledge was obtained indirectly because current transcriptome analysis methods typically require RNA to be converted to complementary DNA (cDNA) before measurements, even though the cDNA synthesis step introduces multiple biases and artefacts that interfere with both the proper characterization and quantification of transcripts. Furthermore, cDNA synthesis is not particularly suitable for the analysis of short, degraded and/or small quantity RNA samples. Here we report direct single molecule RNA sequencing without prior conversion of RNA to cDNA. We applied this technology to sequence femtomole quantities of poly(A)(+) Saccharomyces cerevisiae RNA using a surface coated with poly(dT) oligonucleotides to capture the RNAs at their natural poly(A) tails and initiate sequencing by synthesis. We observed transcript 3' end heterogeneity and polyadenylated small nucleolar RNAs. This study provides a path to high-throughput and low-cost direct RNA sequencing and achieving the ultimate goal of a comprehensive and bias-free understanding of transcriptomes.

An Inside Job for siRNAs

Among the three main categories of small silencing RNAs in insects and mammals-siRNAs, miRNAs, and piRNAs-siRNAs were thought to arise primarily from exogenous sources, whereas miRNAs and piRNAs arise from endogenous loci. Recent work in flies and mice reveals several classes of endogenous siRNAs (endo-siRNAs) that contribute to functions previously reserved for miRNAs and piRNAs, including gene regulation and transposon suppression.

Purification and Concentration of Nucleic Acids

AbstractThe purification and concentration of nucleic acids have become routine procedures in most biology and molecular biology laboratories. This unit covers the basic principles and procedures for the isolation, purification, and manipulation of solutions of DNA or RNA. The basic DNA protocol, using phenol extraction and ethanol precipitation, is appropriate for the purification of DNA from small volumes (&lt;0.4 ml) at concentrations ≤1 mg/ml. Purification of DNA using commercially available silica membrane spin columns is presented as an alternate protocol. Isolation and purification of RNA from mammalian cells or tissues is also examined. Use of the protein denaturant guanidine thiocyanate and water‐saturated phenol, followed by concentration by isopropanol precipitation, for producing small samples of RNA is illustrated in the basic RNA protocol.

Analysis of Affymetrix GeneChip® Data Using Amplified RNA

When small biological samples are collected by microdissection or other methods, amplification techniques are required to provide sufficient target for hybridization to expression arrays. One such technique is to perform two successive rounds of T7-based in vitro transcription. However the use of random primers, required to regenerate cDNA from the first round of transcription, results in shortened copies of cDNA from which the 5' end is missing. In this paper we describe an experiment designed to compare the quality of data obtained from labeling small RNA samples using the Affymetrix Two-Cycle Eukaryotic. Target Labeling procedure to that of data obtained using the One-Cycle Eukaryotic Target Labeling protocol. We utilized different preprocessing algorithms to compare the data generated using both labeling methods and present a new algorithm that improves upon existing ones in this setting.

Optimization and validation of small quantity RNA profiling for identifying TNF responses in cultured human vascular endothelial cells

Introduction Affymetrix oligonucleotide microarrays are widely used in basic and applied research ( Lander, E. S., (1999). Array of hope. Nature Genetics 21, 3–4.; Lockhart, D. J. & Winzeler, E. A., (2000). Genomics, gene expression and DNA arrays. Nature 405, 827–836.). The need for a significant amount of starting RNA has limited its use in applications where the amount of RNA is limiting, such as with Laser Captured Microdissection (LCM), small biopsies, or peripheral blood in rodent models. To overcome this limitation, various RNA amplification and labeling methods have been described, however, further optimization and validation of these methods are needed. Methods Here we reported using the Arcturus technology to optimize amplification and labeling of small amounts of RNA for Affymetrix microarray studies. We assessed the technical feasibility and variation introduced by differences in starting RNA quantity and differences in technical performance by microarray hybridization. Results We demonstrated that the current approach is reliable to amplify as little as 40 ng total RNA, and it is suitable for Affymetrix studies yielding satisfactory quantitative chip performance. We also showed that differences in labeling methods contribute more to variation than the differences in starting RNA quantity per se. As a model, we studied the well-documented TNF-induced inflammatory responses in cultured human vascular endothelial cells. We were able to recapitulate the TNF-induced responses using small RNA sample profiling.

Comparison of sample-labeling techniques in DNA microarray experiments

Usage of DNA microarrays for gene expression analysis has become a common technique in many research laboratories and industry. Several target-labeling techniques have been devised to reduce the amount of RNA required for microarray experiments. In order to facilitate comparison and sharing of microarray data across the laboratories, it is crucial to determine the relative affects of these different sample-labeling techniques on the final results obtained from these experiments. We have compared two labeling methods designed for small RNA samples, an enzyme-based tyramide method (TSA) and a nucleic acid-based dendrimer method, to a more typical direct-labeling method that requires larger amounts of RNA. We observed comparable levels of reproducibility between replicate spots, with all the techniques. The dendrimer method resulted in a minimum number of spots (0.08%) that showed differential labeling due to a bias in the dyes used but resulted in highest background with only 71.4% of the spots measurable (above background) as compared to 93.3% for the TSA technique and 79.7% for the direct-labeling method. The results from differential labeling experiments showed that the dendrimer method performed better than the TSA method in detecting the same set of differentially expressed genes as observed with the direct method. Overall, our results show that the dendrimer method performs better than the TSA method. Differential labeling experiments using the TSA method show a non-linearity in the data at high intensities, leading to skewing of a portion of the data.

Quantitative reverse transcriptase PCR to gauge increased protease-activated receptor 1 (PAR-1) mRNA copy numbers in the Wobbler mutant mouse.

Thrombin acts on cells through the surface protease-activated receptor 1 (PAR-1), a G-protein-coupled member of the seven-transmembrane domain superfamily. On neural cells, thrombin has deleterious effects, killing neurons through apoptosis. Consequently, knowledge of PAR-1 expression in the nervous system may help to elucidate the role of thrombin in neurodegenerative disease. We developed a mimic construct to facilitate the highly sensitive technique of quantitative reverse transcriptase to PCR (qRT-PCR) to measure the differential expression of low copy number PAR-1 mRNA in neurodegenerative model systems. In this article, we report our results comparing homozygous wobbler (wr/wr) mice and normal littermates. By optimizing the transcription and quantitative PCR procedures to facilitate rapid copy number determination in small RNA samples, we documented a fivefold greater level of PAR-1 mRNA in the cervical spinal cord of wr/wr.