Amount Of Total RNA Research Articles

Pretreatment MicroRNA Level and Outcome in Sorafenib-treated Hepatocellular Carcinoma.

Sorafenib represents the first effective targeted therapy for advanced stage hepatocellular carcinoma (HCC); however, adequate patient stratification regarding sorafenib-responsiveness is still missing. Our aim was to analyse the association between the pretreatment microRNA profile of HCC and patient survival under sorafenib treatment. Total RNA was extracted from diagnostic fine-needle aspiration biopsy (FNAB) cytological smears of 20 advanced stage HCC patients collected between June 2008 and July 2012. All patients underwent sorafenib administration after FNA. Clinicopathological and survival data were recorded. Fourteen frequently deregulated miRNAs in HCC (miR-17-5p, miR-18a, miR-21, miR-34a, miR-122, miR-195, miR-210, miR-214, miR-221, miR-222, miR-223, miR-224, miR-140, miR-328) were tested by qRT-PCR. NormFinder software was used to select proper miR (mir-140) as a reference. Satisfactory amount of total RNA was obtained from all the considered samples (mean 10.8 ± 9.3 µg, range 0.2-32.2 µg). Among the analysed miRNAs, high miR-214 expression was associated with smaller tumor size (p=0.019), whereas high miR-17-5p expression correlated with better Eastern Cooperative Oncology Group performance status (p=0.003). The survival analysis revealed that high miR-224 expression was associated with increased progression-free and overall survival (PFS p=0.029; OS p=0.012). Pretreatment microRNA profiling, especially miR-224 expression, might serve as an ancillary tool for the better assessment of expected survival rates for patients under sorafenib treatment.

C-kit/SCF autocrine loop in human NK cells (CCR5P.254)

Abstract Expression of c-kit receptor on CD56bright subset of NK cells has been previously reported. Based on the fact that c-kit receptor and its ligand - stem cells factor (SCF), are co-expressed on many cell types, we have tested the hypothesis that c-kit and SCF might be co-expressed on human peripheral blood NK cells using RT-PCR, flow cytometry and DNA sequencing. NK cells were harvested using the immunomagnetic NK isolation kit and their purity was confirmed by flow cytometry. Analysis of RT-PCR data revealed the presence of two DNA fragments (about 330 bp and 420 bp) in NK cells. Identity of PCR products was directly confirmed by sequencing. To determine the specificity of SCF transcript formation only in c-kit-positive cell subset, NK cells were separated on c-kit-positive and c-kit-negative subsets using CD117 microbeads. No SCF mRNA expression was found in c-kit-negative NK cells even after increasing the quantity of total RNA to 2 μg and the number of amplification cycles to 40. Intracellular flow cytometry analysis with specific anti-SCF antibodies confirmed the expression of SCF protein in human NK cells. Thus, our results provide a definite proof of the existence of c-kit/SCF autocrine loop in c-kit-positive subset of human peripheral blood NK cells. The verification of the functional significance of this new phenomenon is in progress in our laboratory.

697 Novel Approach Reveals That Genetic Variation in/Near FXR/RXR Pathway Genes Predisposes to Nonalcoholic Fatty Liver Disease in Humans

Background and Aim: Free fatty acid-induced hepatocyte cytotoxicity is deeply associated withmorbidity of Non-alcoholic fatty liver disease (NAFLD) (Malhi et al, 2008, Gastroenterology). Saturated free fatty acids induce hepatocyte lipoapoptosis via endoplasmic reticulum stress (ER) response, which leads to translational activation of DR5 and BH3 only protein Bim (Cazanave et al, JBC, 2011). We previously reported that X-box binding protein-1 (XBP-1) is activated downstream of IRE-1 arm of ER stress by free fatty acid treatment upon its splicing (Akazawa et al, 2009, J Hepatol). However, role of XBP-1 during hepatocyte lipoapoptosis remains relatively unexplored. We examined the impact of inhibiting XBP-1 on hepatocyte lipoapoptosis and steatosis employing Toyocamycin, which was recently reported to inhibit ER stress-mediated XBP-1 splicing (Ri M et al, Blood Cancer J. 2012). Methods: We employed hepatocellular carcinoma cell line, Huh-7 cells, for these studies. Cells were incubated with palmitate (200~800μM) in presence or absence of Toyocamycin (0.1~3μM). Apoptosis was assessed by DAPI staining and caspase 3/7 activity assay. Steatosis was assessed by Nile red staining. mRNA expression of Bim and DR5 was examined by real time PCR. Expression of Bim protein and phosphorylation of JNK was assessed by immunoblotting. Results: As expected, Toyocamycin inhibited palmitate-mediated XBP-1 splicing without affecting amount of total RNA. Toyocamycin significantly reduced palmitateinduced hepatocyte lipoapoptosis (p<0.05) and caspase 3/7 activity (p<0.05) in dose dependent manner. Interestingly, Toyocamycin also attenuated free fatty acid-induced steatosis. In addition, Toyocamycin attenuated expression of Bim protein andmRNA (p<0.05) following palmitate treatment. Furthermore, PAmediated-transcriptional activation of DR5was reduced by Toyocamycin (p<0.05). In contrast, palmitate-induced JNK phosphorylation, which also occurs downstream of IRE-1 arm of ER stress, was not inhibited by Toyocamycin. Conclusions: These data imply that Toyocamycin attenuates hepatocyte lipotoxicity as well as steatosis, likely through inhibition of XBP-1 splicing. Toyocamycin might be beneficial for NASH treatment.

696 XBP-1 Inhibitor Toyocamycin Attenuates Palmitate-Induced Hepatocyte Apoptosis and Steatosis

Background and Aim: Free fatty acid-induced hepatocyte cytotoxicity is deeply associated withmorbidity of Non-alcoholic fatty liver disease (NAFLD) (Malhi et al, 2008, Gastroenterology). Saturated free fatty acids induce hepatocyte lipoapoptosis via endoplasmic reticulum stress (ER) response, which leads to translational activation of DR5 and BH3 only protein Bim (Cazanave et al, JBC, 2011). We previously reported that X-box binding protein-1 (XBP-1) is activated downstream of IRE-1 arm of ER stress by free fatty acid treatment upon its splicing (Akazawa et al, 2009, J Hepatol). However, role of XBP-1 during hepatocyte lipoapoptosis remains relatively unexplored. We examined the impact of inhibiting XBP-1 on hepatocyte lipoapoptosis and steatosis employing Toyocamycin, which was recently reported to inhibit ER stress-mediated XBP-1 splicing (Ri M et al, Blood Cancer J. 2012). Methods: We employed hepatocellular carcinoma cell line, Huh-7 cells, for these studies. Cells were incubated with palmitate (200~800μM) in presence or absence of Toyocamycin (0.1~3μM). Apoptosis was assessed by DAPI staining and caspase 3/7 activity assay. Steatosis was assessed by Nile red staining. mRNA expression of Bim and DR5 was examined by real time PCR. Expression of Bim protein and phosphorylation of JNK was assessed by immunoblotting. Results: As expected, Toyocamycin inhibited palmitate-mediated XBP-1 splicing without affecting amount of total RNA. Toyocamycin significantly reduced palmitateinduced hepatocyte lipoapoptosis (p<0.05) and caspase 3/7 activity (p<0.05) in dose dependent manner. Interestingly, Toyocamycin also attenuated free fatty acid-induced steatosis. In addition, Toyocamycin attenuated expression of Bim protein andmRNA (p<0.05) following palmitate treatment. Furthermore, PAmediated-transcriptional activation of DR5was reduced by Toyocamycin (p<0.05). In contrast, palmitate-induced JNK phosphorylation, which also occurs downstream of IRE-1 arm of ER stress, was not inhibited by Toyocamycin. Conclusions: These data imply that Toyocamycin attenuates hepatocyte lipotoxicity as well as steatosis, likely through inhibition of XBP-1 splicing. Toyocamycin might be beneficial for NASH treatment.

RNA-seq Analysis of Early Hepatic Response to Handling and Confinement Stress in Rainbow Trout

Fish under intensive rearing conditions experience various stressors which have negative impacts on survival, growth, reproduction and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and aquaculture production efficiency. In this study, we used RNA-seq to identify transcripts which are differentially expressed in the rainbow trout liver in response to handling and confinement stress. These stressors were selected due to their relevance in aquaculture production. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to a 3 hour handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted to obtain a total of 491,570,566 reads which were mapped onto the previously generated stress reference transcriptome to identify 316 differentially expressed transcripts (DETs). Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (R2 = 0.88). Several gene ontology terms including transcription factor activity and biological process such as glucose metabolic process were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database.Accession NumbersRaw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881.Customized Perl ScriptsAll customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author.

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Transcriptome analysis in maritime pine using laser capture microdissection and 454 pyrosequencing

Maritime pine (Pinus pinaster Aiton) is one of the most advanced conifer models for genomics research. Conifer genomes are extremely large and major advances have recently been made in the characterization of transcriptomes. The combination of laser capture microdissection (LCM) and next-generation sequencing is a powerful tool with which to resolve the entire transcriptome of specific cell types and tissues. In the current work, we have developed a protocol for transcriptomic analyses of conifer tissue types using LCM and 454 pyrosequencing. Tissue sections were isolated using non-fixed flash-frozen samples processed by LCM. Complementary DNA synthesis and amplification from tiny amounts of total RNA from LCM samples was performed using an adapted protocol for C: onifer R: NA A: mplification (CRA+). The cDNA amplification yield and cDNA quality provided by CRA+ were adequate for 454 pyrosequencing. Furthermore, read length and quality results of the 454 runs were near the optimal parameters considered by Roche for transcriptome sequencing. Using the CRA+ protocol, non-specific amplifications were prevented, problems derived from poly(A:T) tails in the 454 sequencing technology were reduced, and read length and read number considerably enhanced. This technical approach will facilitate global gene expression analysis in individual tissues of conifers and may also be applied to other plant species.

Efficient oil palm total RNA extraction with a total RNA extraction kit.

Oil palm tissues are rich in polyphenols, polysaccharides and secondary metabolites; these can co-precipitate with RNA, causing problems for downstream applications. We compared two different methods (one conventional and a kit-based method - Easy-Blue(TM) Total RNA Extraction Kit) to isolate total RNA from leaves, roots and shoot apical meristems of tissue culture derived truncated leaf syndrome somaclonal oil palm seedlings. The quality and quantity of total RNA were compared through spectrophotometry and formaldehyde gel electrophoresis. The specificity and applicability of the protocols were evaluated for downstream applications, including cDNA synthesis and RT-PCR analysis. We found that the conventional method gave higher yields of RNA but took longer, and it was contaminated with genomic DNA. This method required extra genomic DNA removal steps that further reduced the RNA yield. The kit-based method, on the other hand, produced good yields as well as well as good quality RNA, within a very short period of time from a small amount of starting material. Moreover, the RNA from the kit-based method was more suitable for synthesizing cDNA and RT-PCR amplification than the conventional method. Therefore, we conclude that the Easy-BlueTM Total RNA Extraction Kit method is suitable and superior for isolation of total RNA from oil palm leaf, root and shoot apical meristem.

Accuracy of allele frequency estimation using pooled RNA‐Seq

For nonmodel organisms, genome-wide information that describes functionally relevant variation may be obtained by RNA-Seq following de novo transcriptome assembly. While sequencing has become relatively inexpensive, the preparation of a large number of sequencing libraries remains prohibitively expensive for population genetic analyses of nonmodel species. Pooling samples may be then an attractive alternative. To test whether pooled RNA-Seq accurately predicts true allele frequencies, we analysed the liver transcriptomes of 10 bank voles. Each sample was sequenced both as an individually barcoded library and as a part of a pool. Equal amounts of total RNA from each vole were pooled prior to mRNA selection and library construction. Reads were mapped onto the de novo assembled reference transcriptome. High-quality genotypes for individual voles, determined for 23,682 SNPs, provided information on 'true' allele frequencies; allele frequencies estimated from the pool were then compared with these values. 'True' frequencies and those estimated from the pool were highly correlated. Mean relative estimation error was 21% and did not depend on expression level. However, we also observed a minor effect of interindividual variation in gene expression and allele-specific gene expression influencing allele frequency estimation accuracy. Moreover, we observed strong negative relationship between minor allele frequency and relative estimation error. Our results indicate that pooled RNA-Seq exhibits accuracy comparable with pooled genome resequencing, but variation in expression level between individuals should be assessed and accounted for. This should help in taking account the difference in accuracy between conservatively expressed transcripts and these which are variable in expression level.

Discovery, identification and sequence analysis of RNAs selected for very short or long poly A tail in immature bovine oocytes

A major challenge in applying genomics to oocyte physiology is that many RNAs are present but will not be translated into proteins, making it difficult to draw conclusions from RNAseq and array data. Oocyte maturation and early embryo development rely on maternal storage of specific RNAs with a short poly(A) tail, which must be elongated for translation. To resolve the role of key genes during that period, we aimed to characterize both extremes of mRNA: deadenylated RNA and long polyA tails mRNA population in immature bovine oocytes. Using magnetic beads coupled to oligodT, we isolated deadenylated (A-, 20-50 adenosines) from polyadenylated (A+, up to 200 adenosines) RNAs. After transcriptomic analysis, we observed that A+ candidates are associated with short-term processes required for immediate cell survival (translation or protein transport) or meiotic resumption, while several A- candidates are involved in processes (chromatin modification, gene transcription and post-transcriptional modifications) that will be extremely important in the development of the early embryo. In addition to a list of candidates probably translated early or late, sequence analysis revealed that cytoplasmic polyadenylation element (CPE) and U(3)GU(3) were enriched in A- sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U(5)CU(2)) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U(3)GU(3) sequences in bovine but also in zebrafish and Xenopus tropicalis. To further validate our methodology, we measured specific tail length of known candidates (AURKA, PTTG1, H2A1) but also determined the poly(A) tail length of other candidate RNAs (H3F3A, H1FOO, DAZAP2, ATF1, ATF2, KAT5, DAZL, ELAVL2). In conclusion, we have reported a methodology to isolate deadenylated from polyadenylated RNAs in samples with small total RNA quantities such as mammals. Moreover, we identified deadenylated RNAs in bovine oocytes that may be stored for the long-term process of early embryo development and described a conserved motif enriched in the 3'UTR of deadenylated RNAs.

Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice

Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders.

Fidelity and Representativeness of Two Isothermal Multiple Displacement Amplification Systems to Preamplify Limiting Amounts of Total RNA

In this study we investigated the fidelity and representativeness of two novel multiple displacement amplification (MDA) protocols leading to whole transcriptome amplification (WTA). WTA is used to amplify a limiting amount of experimental RNA, allowing its use in downstream applications. Using Phi29 and Bst DNA polymerase-based MDA, henceforth referred to as WTA-Phi and WTA-Bst, respectively, we successfully amplified very low amounts of linearly concatenated cDNA originating from 10 to 100ng of starting RNA. The average yield obtained from 10ng was 3.5 and 4.7μg for WTA-Phi and WTA-Bst, respectively, while 100ng of starting RNA yielded 7.0 and 12.4μg for WTA-Phi and WTA-Bst, respectively. Representational distortion of the templates, analyzed via conventional PCR, showed robust amplification of 11 different transcripts when either WTA-Phi or WTA-Bst synthesized templates were used, while some transcripts were not detected from unamplified templates. Loci representation, a measure of amplification consistency, was evaluated using TaqMan RT-qPCR amplification of five different transcripts, yielding values ranging from 96.4 to 189.3%, comparable to those obtained using genomic target-based MDA systems. The two MDA protocols described in this study efficiently lead to representative WTA, using as little as 10ng of starting RNA.

Expression dynamics of pluripotency genes in chicken primordial germ cells before and after colonization of the genital ridges

Mammalian species utilize an inductive mechanism of germ cell specification, diverting the fate of some of somatic cells toward pluripotency and germ-cell totipotency. It is not known if avian species utilize a similar mechanism nor if, analogous to mammalian primordial germ cells (PGCs), pluripotency genes are continuously upregulated in migrating and genital ridge-colonizing avian PGCs. Thus, this study was conducted to quantify and to analyze the expression profile of pluripotency genes at different stages of chicken PGCs development at Hamburger and Hamilton (HH) stage 14, when the majority of PGCs have entered into the bloodstream; at HH stage 18, when PGCs have resided for 8-12 hr in the bloodstream; and at HH stage 28, when the majority of PGCs are found in the genital ridge. The transcription for Oct4, Sox2, and Nanog continuously decreased from HH stage 14 to HH stage 28. In addition, equal amounts of total RNA could be isolated from chicken PGCs at each stage of development, indicating that the observed drop of transcription of pluripotency genes is not a consequence of transcriptional repression in general. Decreased expression for all three proteins was also observed at HH stage 28. Furthermore, in comparison to blood PGCs, those residing in the gonad have lost their full capacity for colony formation. Our results indicate that, in contrast to mammalian PGCs, chicken PGCs continuously downregulate the expression of pluripotency genes and show a progressive loss of pluripotency-associated features during different stages of germ-line migration.

Variation of Expression Levels of Seven Housekeeping Genes at Different Life-History Stages in Porphyra yezoensis

In order to identify the optimal internal control for relative real-time PCR when studying target gene expression in the red alga Porphyra yezoensis, we quantified the expression of seven housekeeping genes (18S ribosomal RNA, 30S ribosomal protein S8, Polyubiquitin-2, Glyceraldehyde-3-phosphate dehydrogenase, Elongation factor 1-alpha, Beta-tubulin and Actin 3) at different life-history stages. Absolute quantification was done by normalization to total RNA quantity and by normalization to genomic DNA quantity. We used these two normalization approaches, comparing the differences of expression levels of all candidate housekeeping genes between any two generations and across three life-history stages (filamentous sporophytes, leafy gametophytes and conchospores). We found GAPDH had the best stability in all cases and we recommend that GAPDH be considered as a potential internal control for gene expression studies at different life-history stages in P. yezoensis.

Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina's TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.

Regulation of Postsynaptic Retrograde Signaling by Presynaptic Exosome Release

Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling.

Detection of MicroRNAs in Archival Cytology Urine Smears

MicroRNAs’ dysregulation and profiling have been demonstrated to be clinically relevant in urothelial carcinoma (UC). Urine cytology is commonly used as the mainstay non-invasive test for secondary prevention and follow-up of UC patients. Ancillary tools are needed to support cytopathologists in the diagnosis of low-grade UC. The feasibility and reliability of microRNAs profiling by qRT-PCR analysis (miR-145 and miR-205) in archival routine urine cytology smears (affected by fixation/staining [Papanicolau] and room temperature storage) was tested in a series of 15 non-neoplastic and 10 UC urine specimens. Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered. Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06–4.60 µg). Twenty nanograms of total RNA have been calculated to be the minimal total RNA concentration for reliable and reproducible miRNAs expression profiling analysis of archival cytological smears (slope = -3.4084; R-squared = 0.99; efficiency = 1.94). miR-145 and miR-205 were significantly downregulated in UC samples in comparison to non-tumor controls. These findings demonstrate that urine archival cytology smears are suitable for obtaining high-quality RNA to be used in microRNAs expression profiling. Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.

Validation of Internal Reference Genes for Real-Time Quantitative Polymerase Chain Reaction Studies in the Tick,Ixodes scapularis(Acari: Ixodidae)

Obtaining reliable gene expression data using real-time quantitative polymerase chain reaction (qPCR) is highly dependent on the choice of normalization method. We tested the expression stability of multiple candidate genes in the salivary glands (SG) and synganglia (SYN) of female Ixodes scapularis (Say) ticks in multiple blood-feeding phases. We found that the amount of total RNA in both the SG and SYN increases dramatically during tick feeding, with 34x and 5.8x increases from 62 and 7.1 ng of unfed tick, respectively. We tested candidate genes that were predicted from I. scapularis genome data to encode glyceraldehyde 3-phosphate dehydrogenase (gapdh), ribosomal protein L13A (l13a), TATA box-binding protein (tbp), ribosomal protein S4 (rps4), glucose 6-phosphate dehydrogenase (gpdh), and beta-glucuronidase (gusb). The geNorm and NormFinder algorithms were used to analyze data from different feeding phases (i.e., daily samples from unfed to fully engorged females over a 7-d period in three replicate experiments). We found that the rps4 and l13a genes showed highly stable expression patterns over the feeding duration in both the SG and SYN. Furthermore, the highly expressed rps4 gene makes it useful as a normalization factor when we perform studies using minute amounts of dissected tissue for qPCR. We conclude that rps4 and l13a, whether individually or as a pair, serve as suitable internal reference genes for qRT-PCR studies in the SG and SYN of I. scapularis.

Suprachiasmatic Nucleus Neuropeptide Expression in Patients with Huntington's Disease

To study whether sleep and circadian rhythm disturbances in patients with Huntington's disease (HD) arise from dysfunction of the body's master clock, the hypothalamic suprachiasmatic nucleus. Postmortem cohort study. Eight patients with HD and eight control subjects matched for sex, age, clock time and month of death, postmortem delay, and fixation time of paraffin-embedded hypothalamic tissue. Using postmortem paraffin-embedded tissue, we assessed the functional integrity of the suprachiasmatic nucleus in patients with HD and control subjects by determining the expression of two major regulatory neuropeptides, vasoactive intestinal polypeptide and arginine vasopressin. Additionally, we studied melatonin 1 and 2 receptor expression. Compared with control subjects, the suprachiasmatic nucleus contained 85% fewer neurons immunoreactive for vasoactive intestinal polypeptide and 33% fewer neurons for arginine vasopressin in patients with HD (P = 0.002 and P = 0.027). The total amount of vasoactive intestinal polypeptide and arginine vasopressin messenger RNA was unchanged. No change was observed in the number of melatonin 1 or 2 receptor immunoreactive neurons. These findings indicate posttranscriptional neuropeptide changes in the suprachiasmatic nucleus of patients with HD, and suggest that sleep and circadian rhythm disorders in these patients may at least partly arise from suprachiasmatic nucleus dysfunction.

Expression analysis and identification of antimicrobial peptide transcripts from six North American frog species

Frogs secrete antimicrobial peptides onto their skin. We describe an assay to preserve and analyze antimicrobial peptide transcripts from field-collected skin secretions that will complement existing methods for peptide analysis. We collected skin secretions from 4 North American species in the field in California and 2 species in the laboratory. Most frogs appeared healthy after release; however, Rana boylii in the Sierra Nevada foothills, but not the Coast Range, showed signs of morbidity and 2 died after handling. The amount of total RNA extracted from skin secretions was higher in R. boylii and R. sierrae compared to R. draytonii, and much higher compared to Pseudacris regilla. Interspecies variation in amount of RNA extracted was not explained by size, but for P. regilla it depended upon collection site and date. RNA extracted from skin secretions from frogs handled with bare hands had poor quality compared to frogs handled with gloves or plastic bags. Thirty-four putative antimicrobial peptide precursor transcripts were identified. This study demonstrates that RNA extracted from skin secretions collected in the field is of high quality suitable for use in sequencing or quantitative PCR (qPCR). However, some species do not secrete profusely, resulting in very little extracted RNA. The ability to measure transcript abundance of antimicrobial peptides in field-collected skin secretions complements proteomic analyses and may provide insight into transcriptional mechanisms that could affect peptide abundance.