RNA Purity Research Articles

Automated nucleic acids purification from fecal samples on a microfluidic cartridge

In this paper, we describe the development of a microfluidic cartridge and its controlling device for fully automated nucleic acid purification from fecal samples. All steps of the standard complicated process, including fecal sample pretreatment, lysis, washing, and elution, were integrated into a microfluidic cartridge that could be operated with minimal hands-on time requirement. The performance of the microfluidic cartridge was evaluated using fecal samples spiked with bacteria or virus as well as fecal microbiome analysis. Microfluidic cartridge-based nucleic acid purification exhibited similar or even higher performance compared to commercialized products with respect to the concentration and purity of bacterial DNA or viral RNA. It also showed a similar or higher detection capa city for microbiome diversity in clinical fecal samples, as well as similar or higher extraction efficiency from such samples. The development of microfluidic cartridge-based fully automated platform for nucleic acid purification from fecal samples may be an important milestone towards developing a point-of-care test for rapid and simple diagnosis of gastroenteritis and gut microbiome analysis.

Deregulated Expression of Long Non-Coding RNAs UCA1, NEAT1 and BDNF-As in Multiple Myeloma

Introduction. Multiple myeloma (MM) is the second most common hematological malignancy in the world. It is characterized by increasing rate of various genetic mutations and dysregulated pathways. This work aims to find out if this phenomenon is also reflected in dysregulation of the so-called long non-coding RNA molecules (lncRNA). These molecules are over 200 nt long and primarily localized in the nucleus. It seems increasingly obvious that lncRNAs play a crucial role in human diseases and during hematopoiesis. Presumably, lncRNA affect hematological malignancies including MM by regulating the expression of oncogenes, tumor suppressor and key factors involved in hematopoiesis. We identified a disease-specific cellular lncRNA signature using a cohort of MM patients in comparison to healthy donors (HD).Methods. Fifty CD138+ samples obtained from newly diagnosed MM patients and HD were evaluated for this study. Total RNA was extracted from MM cells using miRNeasy Mini Kit or miRNeasy Micro Kit (all Qiagen) according to the manufacturer’s instructions. Concentration and purity of RNA were determined spectrophotometrically by NanoDrop ND-1000 (Thermo Scientific, USA). Screening analysis of 83 lncRNA was performed on 6 MM patients and 6 HD using RT2 lncRNA PCR Array - Human lncRNA Finder (Qiagen). Significantly deregulated lncRNAs between MM vs HD were validated by qPCR using relative quantification approach 2-ΔCt on a larger cohort of patients and HD. Briefly, High-capacity cDNA reverse transcription kit (Applied Biosystem, USA), was used to synthesize cDNA from 200 ng RNA according to the manufacturer’s recommendations. Expression levels of ZFAS1, UCA1, BDNF-AS, NEAT1 and FAS-AS1 were detected by RT-qPCR using TaqMan non-coding RNA assay (ZFAS1: Hs01379985_m1, FAS-AS1: Hs04233476_s1, BDNF-AS: Hs01010228_m1, UCA1: Hs01909129_s1, NEAT1: Hs03453535_s1), expression level of GAPDH using Human GAPD (GAPDH) Endogenous Control (VIC®/MGB probe, primer limited) and TaqMan Gene Expression Master Mix (all Applied Biosystem, USA). qPCR was performed using the Applied Biosystem 7500 Sequence Detection System. Analysis of the RT-qPCR data was performed using SDS version 2.0.1 software (Applied Biosystem, USA). Expression data from lncRNAs profiling were statistically evaluated in the environment of statistical language R by use of Bioconductor package and LIMMA approach combined with hierarchical clustering (HCL). P values were adjusted according to Bonferroni correction for multiple comparisons. Statistical differences between lncRNAs expression levels in MM patients and HD were evaluated by non-parametric Mann-Whitney U test. Receiver Operating Characteristic (ROC) analysis was used to calculate specificity and sensitivity of each lncRNA. P values <0.05 were considered significant.Results.RT2 lncRNA PCR Array profiling revealed 27 deregulated lncRNAs (all p<0.01) between MM patients and HD. ZFAS1, UCA1, BDNF-AS, NEAT1 and FAS-AS1 expression was further verified on a larger cohort of MM and HD samples. As the difference in ZFAS1 and FAS-AS1 expression between MM and HD was not significant (p=0.7; p=0.2, respectively), it was excluded from further analyses. UCA1 was significantly down-regulated (p<0.0001), NEAT1 and BDNF-AS were up-regulated in MM samples when compared with HD (both p<0.00000001). To discriminate MM from HD, receiver operating characteristic (ROC) curve was calculated. It revealed sensitivity of 100% (95%CI: 91.6 - 100.0) and specificity of 100% (95%CI: 63.1 - 100.0), and area under curve (AUC) = 1.000 for both, NEAT1 and BDNF-AS lncRNA expression and sensitivity of 95.24% (95%CI: 83.8 - 99.4) and specificity of 75.00% (95%CI: 34.9 - 96.8), and area under curve (AUC) = 0.905 for UCA1 expression levels (Fig. 1). We suppose that these dysregulated lncRNAs could have a biological relevance in MM since BDNF-AS is an antisense RNA for BDNF, a significant stimulating factor of osteoclasts in MM, NEAT1 and UCA1 were described as dysregulated in several types of cancer, including hematological malignancies. Further validations are currently ongoing.Conclusions. Altogether, our first observations demonstrate that cellular lncRNA UCA1, NEAT1 and BDNF-AS may be involved in pathophysiological processes occurring in MM cells and prompt further studies in this field.Grant support: AZV 15-29508A [Display omitted] DisclosuresHajek:Onyx: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Novartis: Research Funding.

Extraction of high-quality RNA from human articular cartilage

Extracting high-quality RNA from articular cartilage is challenging due to low cellularity and high proteoglycan content. This problem hinders efficient application of RNA sequencing (RNA-seq) analysis in studying cartilage homeostasis. Here we developed a method that purifies high-quality RNA directly from cartilage. Our method optimized the collection and homogenization steps so as to minimize RNA degradation, and modified the conventional TRIzol protocol to enhance RNA purity. Cartilage RNA purified using our method has appropriate quality for RNA-seq experiments including an RNA integrity number of ∼8. Our method also proved efficient in extracting high-quality RNA from subchondral bone.

Evaluation of RNA extraction methods in rice and their application in expression analysis of resistance genes against Magnaporthe oryzae

ABSTRACTExtraction of RNA of high quality and integrity is essential for gene expression studies and all downstream RNA-based techniques. The leaves of 16 merit Malaysian rice varieties were used to isolate total RNA using five different methods. The quantity, quality and integrity of extracted RNA were confirmed using three different means. The ratios of A260/280 ranged from 2.12 to 2.20. Electrophoresis (1.5% agarose gel) was performed, illustrating intact and sharp bands representing the 28S, 18S, 5.8S and 5S ribosomal subunits of RNA, presenting intact RNA. RNA quality was verified using semi-quantitative polymerase chain reaction (sqPCR). The objective of this study was to identify different genes involved in the resistance of rice plants using high-quality RNA extracted 31 h after inoculation of Magnaporthe oryzae pathotype P7.2. The expression levels of eight blast resistance genes, Pikh, Pib, Pita, Pi21, Pi9, Os11gRGA8, OsWRKY22 and OsWRKY45, were evaluated by real-time PCR (RT-PCR). Real-time PCR was performed to identify candidate genes using RNA extracted by the TRIzol method, which showed the highest score compared with other methods in terms of RNA quantity, purity and integrity. In addition, the results of real-time PCR confirmed that the up-regulation of seven blast resistance genes may confer stronger resistance for the MR 276 variety against M. oryzae pathotype P7.2.

Effect of a Mushroom (Coriolus versicolor) Based Probiotic on the Expression of Toll-like Receptors and Signal Transduction in Goat Neutrophils

&lt;p class="1Body"&gt;Neutrophils recognize and destroy pathogens through activation of the Toll like receptor (TLR) system as part of the inflammatory response of innate immunity. The expression and modulation of genes in the TLR signaling pathway in caprine blood neutrophils was investigated. Following initial screening for infection, goats (N=15) were assigned to three groups of five (n=5) individuals. Goats were drenched daily with 10 mL of powdered CorPet (Mycology labs Inc) soaked in hot (treatment I) or cold (treatment II) sterile filtered endotoxin free water, for a 4-week period. A control group of five age-matched goats received sterile water (treatment III). Blood was collected weekly and analyzed for packed cell volume and white blood cell differential counts. At weeks 1 and 4 neutrophils were isolated, using differential centrifugation and hypotonic lysis of red blood cells. The concentration and purity of total RNA isolated using Trizol was determined on a Nanodrop spectrophotometer. The RETROscript kit was used to synthesize cDNA. The expression of 84 genes in the human TLR signaling pathway RT&lt;sup&gt;2&lt;/sup&gt; PCR Array was evaluated using real time PCR and the Livak method. The house keeping gene GAPDH was used to normalize the data. At week 1 untreated goats expressed 48 genes in the pathway. Goat neutrophils expressed 10 TLRs. Mushroom extracts modulated expression of and signaling by TLR. These results will help in the definition of the role of TLR expression in neutrophils and its contribution to goat innate immunity. Further this may aid in the design of therapeutics for goat health.&lt;/p&gt;

Stabilization of circulating thyroglobulin mRNA transcripts in patients treated for differentiated thyroid carcinoma

Background The clinical utility of serum thyroglobulin in the follow-up of patients with differentiated thyroid carcinoma may be compromised by the presence of endogenous antithyroglobulin antibodies. To prevent interference by antithyroglobulin antibodies several groups have developed real-time PCR-based assays for quantification of blood thyroglobulin mRNA levels. For accurate quantification of thyroglobulin mRNA in blood preanalytical factors must be recognized and controlled. In this study, we evaluate the effect of different blood RNA stabilizing systems - the Tempus Blood RNA system and the PAXgene Blood RNA system - and storage time on RNA yield and quality, and thyroglobulin mRNA stability. Methods Blood samples from 11 patients previously treated for differentiated thyroid carcinoma were collected in K2-EDTA, Tempus and PAXgene tubes and maintained at room temperature. RNA was isolated following storage for 0 and 72 h, and RNA yield, integrity and purity was determined. Thyroglobulin, GAPDH and ACTB mRNA levels were quantified by semi-quantitative real-time PCR. Results The RNA yield was significantly higher for blood collected in Tempus tubes compared with PAXgene tubes following storage for 72 h at room temperature ( P = 0.0011). High-quality RNA could be extracted from blood collected in PAXgene and Tempus tubes. Blood collected in K2-EDTA tubes, but not in PAXgene and Tempus tubes, showed significant changes in thyroglobulin mRNA levels following storage for 72 h at room temperature ( P = 0.0263). Conclusions Stabilization of blood in PAXgene and Tempus tubes enables storage at room temperature for up to 72 h, without compromising thyroglobulin mRNA levels.

Towards next-generation sequencing analytics for foodborne RNA viruses: Examining the effect of RNA input quantity and viral RNA purity

Detection and identification of viruses in food samples are technically challenging due largely to the low viral copy number in contaminated food items, and the lack of effective culture enrichment methods that are amenable to regulatory applications for many of the common foodborne viruses. Using an Illumina MiSeq platform and two hepatitis A virus (HAV) cell-culture adapted strains as a representative enteric virus species, this study examined the limits of single-stranded RNA (ssRNA) viral detection following next-generation sequencing without pre-amplification of the viral genome. Complete viral genome sequences were obtained from HAV samples of varying purities and with an input as low as 2ng total RNA containing 1.4×105 copies of viral RNA. In addition, single nucleotide variations were reproducibly detected over the range of concentrations examined, and their identity confirmed by alternate sequencing technology. In summary, next-generation sequencing technology has the potential for sensitive detection/identification of a viral genome at a low copy number. This study provides a benchmark for metagenomic sequencing application as is required for virus detection in complex food matrices using a culture-independent diagnostic approach.

RNA quality evaluation of tissue samples in the human brain tumor biological sample bank of the Department of Neurosurgery of Beijing Tiantan Hospital

Objective To evaluate the quality of RNA samples since the establishment of the brain tumor bank of the Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University for 5 years. Methods The brain tumor samples were collected from the Department of Neurosurgery, Tiantan Hospital, Capital Medical University between 2011 and 2015. Twenty samples were randomly selected each year (a total of 100 samples). The total RNA was extracted from tissues by using Trizol reagent, and the RNA purity, concentration, integrity, and GAPDH and ACTB reference gene expression levels in the samples were detected. Results The sample qualification rates of RNA from 2011 to 2015 were 25%, 75%, 85%, 90%, and 100%, respectively. Most of the purity and integrity of the total RNA extracted from the samples in 2011 were poor. RNA had part of the degradation and could not effectively amplify the reference genes GAPDH and ACTB. The Ct value was significantly higher than the mean Ct value of the samples between 2012 and 2015 (P<0.01). The total ratio of A260/A280 of RNA extracted from the samples between 2012 and 2015 reached 1.8 to 2.1, the ratio of A260/A230 could reach more than 2.0, the integrity of RNA in most of the samples was better, and no degradation was observed. Eighty samples could be used to conduct the effective amplification of GAPDH and ACTB genes. Conclusions When conducting the experiment study of the RNA levels, using the samples of the storage time within 4 years is recommended, the quality detection of RNA should be conducted before using the tissue samples, and the sample quality should be monitored regularly each year. The quality evaluation should be conducted before leaving the sample bank. Key words: Brain neoplasms; Biological sample bank; RNA; Quality evaluation

Micro-RNAs as positive biomarkers of pathological complete response after neoadjuvant chemoradiotherapy in locally advanced rectal carcinoma.

624 Background: Neoadjuvant chemoradiotherapy (nCRT) follow by surgery, as treatment for locally advanced rectal carcinoma (LARC), has improve local disease control, increase of complete pathological response (cPR) and preserve anal sphincter. cPR is associated with better prognosis. CRT act mainly through induction of DNA damage. MicroRNAs (miRNAs) are small non-coding RNAs able to regulate gene expression at post-transcriptional level. miRNAs are involved in the regulation of DNA damage/repair mechanism. Our goal was to measure expression of miRNAs involved in DNA damage/repair genes and correlated with cPR. Methods: Retrospectively, we analyzed the initial paraffin embedded tissue block of 20 Mexican patients with LARC treated with nCRT at National Cancer Institute of Mexico between 2010-2013. Treatment response was evaluated with Ryan Classification. Ten patients had cPR (Ryan Grade 0) meanwhile other 10 poor response (Ryan Grade 3). RNA extraction was done with RNeasy FFPE (Qiagen) Kit. RNA concentration and purity was assessed using NanoDrop 2000 Spectrophotometer. miRNAs expression were evaluated by real-time polymerase chain reaction (PCR) analysis with TaqMan Probes from Applied Biosystems. Statistical analysis was carried out with IBM SPSS Statistics 22.0. Differential expression between the two groups was performed with Chi square test. Results: There wasn’tsignificant difference betweenclinical/demographic features between the 2 groups. All patients received CRT at a total dose of 4500 cGy of pelvic irradiation, concomitantly with fluoropyrimidine. Surgical treatment was performed 14 weeks after completion of nCRT. The 2 miRNAs (188-5p and 590-5p) was overexpressed in the cPR group vs poor response group (p = 0.011 and p = 0.057, respectively). Conclusions: We evaluated the expression of miR590-5p and miR188-5p because they are related to DNA repair genes such as: MSH2, ERCC3, BRCC3 and XRCC5. We found overexpression in both miRNAs. Even though miR590 5p didn't reach statistical significance this maybe because of simple size.Our results now are been analyzed with a biological and functional genes network platform: Ingenuity Pathway Analysis.

Monitoring of Chicken RNA Integrity as a Function of Prolonged Postmortem Duration.

Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect A260/A280 and A260/A230 (p≥0.05), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples (p≥0.05), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.

Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer

Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell–cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines.

Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

BackgroundThe complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples.ResultsComparison of three commercially available RNA extraction kits with the method of Zoetendal revealed that the Powermicrobiome Kit (MoBio) performed best with respect to RNA yield and purity. Next, the influence of the stabilization reagent during sample storage for up to 15 days was studied. RIN analysis and qRT-PCR of spiked-in and indigenous genes revealed that RNA Later preserved mRNA integrity most efficiently, while samples conserved in RNA Protect showed substantial mRNA decay. Using the optimized pipeline developed here, recovery rates for spiked-in E.coli cells expressing fluorescing proteins were 8.7-9.7 % for SuperfolderGFP and 14.7-17.8 % for mCherry. The mRNA of stabilized stool samples as well as of snap-frozen controls was sequenced with Illumina Hiseq, yielding on average 74 million reads per sample. PCoA analysis, taxonomic classification using Kraken and functional classification using bwa showed that the transcriptomes of samples conserved in RNA Later were unchanged for up to 6 days even at room temperature, while RNA Protect was inefficient for storage durations exceeding 24 h. However, our data indicate that RNA Later introduces a bias which is then maintained throughout storage, while RNA Protect conserved samples are initially more similar to the snap frozen controls. RNA Later conserved samples had a reduced abundance of e.g. Prevotellaceae transcripts and were depleted for e.g. COG category “Carbohydrate transport and metabolism”.ConclusionSince the overall similarity between all stool transcriptional profiles studied here was >0.92, these differences are unlikely to affect global comparisons, but should be taken into account when rare but critically important members of the stool microbiome are being studied.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1694-y) contains supplementary material, which is available to authorized users.

Optimisation of total RNA extraction from bovine oocytes and embryos for gene expression studies and effects of cryoprotectants on total RNA extraction

Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, (GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n = 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7 to 2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng μl(-1) for MTP, against 46.4 ± 9.5 ng μl(-1) from CKP and 476 ± 12.9 ng μl(-1) for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng μl(-1) and 40.0 μ 12.4 ng μ(-1), respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to PROH (1.8 vs 1.2) (p < 0.05). Better results were also observed on the MTP for gene amplification when compared with the other protocols. For gene quantification, the proposed protocol quantified DNMT1 gene with PCR efficiency (0.933) after normalization against GAPDH and SDHA. Amplification and quantification of genes proved specificity and efficiency of the MTP over the other protocols.

Housekeeping genes for studies of plasma microRNA: A need for more precise standardization

Plasma microRNAs (miRNAs) are promising biomarkers for many forms of cancer in humans; however, a fundamental concern is the lack of standardization in current data acquisition and reporting. Part of this problem lies in the use of numerous, different housekeeping genes (HKG) for the acquisition of real-time polymerase chain reaction data. This existing practice of using different HKGs generally is accepted, but reproducibility of data for comparison and validation between different laboratories calls for improvement. The need for data reproducibility standardization is crucial. An ideal plasma HKG (1) should be expressed in all samples, (2) have medium-to-high levels of expression, and (3) have consistently measurable levels of expression. Total RNA was extracted from 200-μL plasma samples via a modified miRNeasy (QIAGEN) extraction technique with yeast carrier. Total RNA purity was assessed with a Nanodrop 2000 spectrophotometer (Thermo Scientific). The cycle threshold (Ct) was fixed at 0.03 for all samples. We investigated 10 potential HKGs based both on reports in the literature and our previous data. The potential HKGs were Let-7a, Let-7d, Let-7g, miR-16, RNU6, RNU48, miR-191, miR-223, miR-484, and miR-520d-5p. Once all samples were run for each potential HKG, the mean Ct and SD was calculated for all sample groups, allowing for comparison among HKGs. We screened 380 miRNAs by using microfluidic array technology (Applied Biosystems) in a discovery cohort of 20 colorectal cancer (CRC) patients, 10 patients each with breast cancer (BC), lung cancer (LC), pancreatic cancer (PC), 11 patients with colorectal adenoma, and 12 controls. The mean Ct and SD was calculated for RNU6, miR-520d-5p, miR-16, miR-191, miR-223, and miR-484, which were expressed in all samples. Let-7a, Let-7d, Let-7g, and RNU48 were only expressed in 26%, 7%, 10%, and 8% of samples, respectively, and therefore were deemed to be insufficiently reliable HKGs. Only miRNAs with >50% expression were included in this statistical analysis. U6 and miR-520d-5p had the most consistent Ct as well as the least SD. The use of both RNU6 and 520d-5p as HKGs provided reliable results. Among HKGs that were expressed in all samples, we suggest that RNU6 and miR-520d-5p were the best candidates for HKGs for studies of plasma miRNA because of the consistent and high Ct in all samples and a very narrow, reproducible SD.

Vasopressin decreases pulmonary-to-systemic vascular resistance ratio in a porcine model of severe hemorrhagic shock.

Vasopressors are gaining renewed interest as treatment adjuncts in hemorrhagic shock. The ideal vasoconstrictor will increase systemic blood pressure without increasing pulmonary vascular resistance (PVR), which hinders pulmonary perfusion and exacerbates hypoxemia. However, the selectivity of pressors for pulmonary versus systemic vasoconstriction during hemorrhage has not been characterized. The purpose of this study was to test the hypothesis that vasopressin (VP) has distinct effects on pulmonary versus systemic hemodynamics, unlike the catecholamine vasopressors norepinephrine (NE) and phenylephrine (PE). Anesthetized and ventilated pigs were assigned to resuscitation with saline only (n = 7) or saline with VP (n = 6), NE (n = 6), or PE (n = 6). Animals were hemorrhaged to a target volume of 30 mL/kg and a mean arterial pressure of 35 mmHg. One hour after the start of hemorrhage, animals were resuscitated with saline up to one shed blood volume, followed by either additional saline or a vasopressor. Hemodynamics and oxygenation were measured hourly for 4 h after the start of hemorrhage. Vasopressin increased systemic vascular resistance (SVR) while sparing the pulmonary vasculature, leading to a 45% decrease in the PVR/SVR ratio compared with treatment with PE. Conversely, NE induced pulmonary hypertension and led to an increased PVR/SVR ratio associated with decreased oxygen saturation. Phenylephrine and crystalloid had no significant effect on the PVR/SVR ratio. Sparing of pulmonary vasoconstriction occurs only with VP, not with administration of crystalloid or catecholamine pressors. The ability of VP to maintain blood oxygenation indicates that VP may prevent hypoxemia in the management of hemorrhagic shock.

Adapted approach to profile genes while reconciling Vegf-a mRNA expression in the developing and injured lung.

During lung development and injury, messenger RNA (mRNA) transcript levels of genes fluctuate over both space and time. Quantitative PCR (qPCR) is a highly sensitive, widely used technique to measure the mRNA levels. The sensitivity of this technique can be disadvantageous and errors amplified when each qPCR assay is not validated. In contrast to other organs, lungs have high RNase activity, resulting in less than optimal RNA integrity. We implemented a strategy to address these limitations in developing and injured lungs. Parameters were established and a filter designed that optimized amplicon length and included or excluded samples based on RNA integrity. This approach was illustrated and validated by measuring mRNA levels including Vegf-a in newborn mouse lungs that were injured by 85% oxygen (hyperoxia) for 12 days and compared with control (normoxia). We demonstrate that, in contrast to contradictory Vegf-a expression when normalized to the least suitable housekeeping genes, application of this filter and normalization to most suitable three housekeeping genes, Hprt, Eef2, and Rpl13a, gave reproducible Vegf-a expression, thus corroborating the sample filter. Accordingly, both short amplicon length and proper normalization to ranked, evaluated genes minimized erroneous fluctuation and qPCR amplification issues associated with nonideal RNA integrity in injured and developing lungs. Furthermore, our work uncovers how RNA integrity, purity, amplicon length, and discovery of stable candidate reference genes enhance precision of qPCR results and utilizes the advantages of qPCR in developmental studies.

Analysis of PTEN and ARID1A expression in Endometriosis disease using RT‐PCR assay

Endometriosis is defined as the presence of endometrial glands and stroma outside of the uterine musculature and endometrial lining. Regulation of gene expression is a flexible, yet stable, mechanism that maintains a cellular memory that could result in a disease state.The aim of this study was to analyse mRNA levels expression in endometrial tissue and endometrium from women with and without endometriosis. We will analyze mRNA transcription activity in PTEN (phosphatase and tensin homolog), and ARID1A (AT rich interactive domain 1A) in fresh tissue from patients with endometriosis using RT‐PCR assay. In this study, we used NCBI database for PTEN and ARID1A transcript. The RNA extraction was performed using commercial kit Qiagen in endometrium and endometriosis fresh tissues. RNA integrity, quality, and purity were stringently analyzed by Experion technology. RT‐PCR analysis showed no consider PTEN and ARID1A mRNA expression in eutopic and ectopic endometrium. The expression levels of PTEN transcript (Exon5 and9) and ARID1A transcript (Exon 9 and 20) by RT‐PCR analysis were observed of mRNA expression of the PTEN and ARID1A in endometriotic tissues vs. controls. We observed that no differences statically between in eutopic and ectopic endometrium patterns of PTEN transcript P value (0.6499) and ARID1A transcript P value (0.9789) in this transcription of target genes. Our study will continue to increase more sample to improve the power analysis of the samples and provide further evidence to different patterns of PTEN and ARID1A transcription of target of these genes in endometriosis to transition to cancer.

Association between tissue miR-141, miR-200c and miR-30b and bladder cancer: a matched case-control study.

To evaluate the expression of microRNAs in tissue samples from patients with bladder cancer and to compare it with healthy adjacent tissue samples as controls. Thirty five tissue samples from patients with newly diagnosed untreated bladder transitional cell carcinoma and 35 adjacent normal urothelium were collected during 2013 to 2014. TRIzol reagent was used to isolate total RNA including microRNAs. RNA concentration and purity were determined using a nanodrop spectrophotometer. Also 1% agarose gel electrophoresis was used to assess integrity of RNA. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) method was performed using the PARSGENOME microRNA RT-PCR system. Data was analyzed by STATA 11. A couple of patients were female the remainder were male. Mean age of patients were 71.06 ± 11.43 years. The expression level of miR-30b, miR-141 and miR-200c in case group were significantly higher than that of control normal tissue samples. miR-141 had higher expression rate in malignant tissue than two other miRNAs (P < .001). There was a more expression rate of miR-200c, miR-141 and miR-30b in bladder cancer tissues than healthy adjacent control tissues. Further studies are needed to draw final conclusion.

Evaluation of DNA/RNAshells for room temperature nucleic acids storage.

Traditional nucleic acids preservation methods rely on maintaining samples in cold environments, which are costly to operate and time sensitive. Recent work validated that using room temperature for the storage of nucleic acids is possible if the samples are completely protected from water and oxygen. Here, we conducted accelerated aging and real-time degradation studies to evaluate the new technology DNAshell and RNAshell, which preserves DNA and RNA at room temperature, including the DNA and RNA yield, purity, and integrity. DNA and RNA solutions are dried in the presence of stabilizers in stainless steel minicapsules, then redissolved after different time points of heating and storing at room temperature. Results show that DNAshell and RNAshell ensure the safe storage of nucleic acids at room temperature for long periods of time, and that the quality of these nucleic acids is suitable for common downstream analysis.

Toward a more efficient isolation of total RNA from loquat (Eriobotrya japonicaLindl.) tissues

Introduction. Loquat (Eriobotrya japonica Lindl.) tissues contain considerable amounts of polyphenolic compounds and polysaccharides which can copurify with RNA. This can render the RNA unsuitable for DNA synthesis and makes the isolation of high quality RNA from loquat tissues particularly difficult. Materials and methods. Total RNA was extracted from leaves, buds and fruit of loquat by adapting the method described by Song et al. in 2011. This involved improvements both to the extraction protocol and RNA purity. Results and discussion. Our modified method resulted in the isolation of high quality RNA with yields 6–20 times higher than that reported for existing methods. We obtained A260/280 ratios of 2–2.40 A260/230 ratios of 2–2.31 and RNA integrity numbers (RIN) of 8 to 8.3. The RNA obtained has reached the requested quality for downstream applications such as quantitative real-time polymerase chain reaction (RT-qPCR) experiments.