Pg Of Total RNA Research Articles

On-Chip Separation and Analysis of RNA and DNA from Single Cells

The simultaneous analysis of RNA and DNA of single cells remains a challenge as these species have very similar physical and biochemical properties and can cross-contaminate each other. Presented is an on-chip system that enables selective lysing of single living cells, extraction, focusing, and absolute quantification of cytoplasmic RNA mass and its physical separation from DNA in the nucleus using electrical lysing and isotachophoresis (ITP). This absolute quantitation is performed without enzymatic amplification in less than 5 min. The nucleus is preserved, and its DNA fluorescence signal can be measured independently. We demonstrate the technique using single mouse lymphocyte cells, for which we extracted an average of 14.1 pg of total RNA per cell. We also demonstrate correlation analysis between the absolute amount of RNA and relative amount of DNA, showing heterogeneity associated with cell cycles. The technique is compatible with fractionation of DNA and RNA and with downstream assays of each.

The structure and role of mammalian sperm RNA: a review

The main role of sperm is the delivery of the paternal genome into the oocyte during fertilisation. However, several lines of evidence have indicated that mammalian spermatozoa contribute more than just their DNA, namely, they also deliver a large range of RNA molecules. Microarray analysis has revealed a complex population of 3000 different kinds of messenger RNA that are delivered to oocytes by sperm and ejaculated spermatozoa are estimated to contain about 0.015 pg of total RNA. Some of the transcripts encode proteins crucial for early embryo development. Messenger RNAs from sperm also help to protect the paternal genes, which have an integral role soon after fertilisation. The molecular participation of the oocyte during fertilisation is well understood but the function of the sperm in this process remains unclear. During spermatogenesis the structure of the male haploid genome is permanently modified. Transition proteins (TNPs), protamines (PRMs) and histones (HILS-spermatid specific linker histone) play a unique role in spermatid chromatin compaction. In this review, the structure and role of sperm RNA as well as chromatin organisation during spermatogenesis are discussed.  

147 HYDROSTATIC PRESSURE STRESS TREATMENT OF MOUSE OOCYTES INFLUENCES PROTEIN SYNTHESIS AT THE 4-CELL STAGE

Sublethal stress treatment [namely hydrostatic pressure (HP)] has been reported to improve the cryotolerance and developmental competence of mammalian gametes and embryos at cryopreservation, in vitro maturation, parthenogenetic activation and somatic cell NT (Pribenszky and Vajta 2011 Reprod. Fertil. Dev. 23, 48–55). In our previous study mouse oocytes were HP stress treated and then vitrified, warmed, fertilized by intracytoplasmic sperm injection (ICSI), cultured and transferred. Blastocyst rate, inner cell mass cell number and birth rate were significantly increased in the treated groups (Pribenszky et al. 2010 Fertil. Steril. 94, S32). In our present study we implemented gene expression microarray experiments to analyse the transcriptome of HP-treated eggs and 4-cell stage embryos developed from these oocytes. At first, 4 pools of 50 mouse oocytes were treated with 20 MPa for 60 min at 37°C in a programmable HP machine (Cryo-Innovation Inc., Budapest, Hungary). Second, mouse oocytes were treated with HP likewise, fertilized by ICSI and cultured to 4-cell stage. The controls were handled identically with no HP treatment. After ICSI, 78% of the treated and 82% of the control oocytes survived the procedure, of which 76 and 65% developed to 4-cell stage; furthermore 3–3 pools of 30 to 44 and 24 to 36 embryos were used for RNA isolation, respectively. The labelled cRNA target generated from 240 pg of total RNA was hybridized to 4 × 44 K whole mouse genome chips (Agilent Technologies, Palo Alto, CA, USA). All the microarray data have been deposited in the NCBI Gene Expression Omnibus database (accession GSE28443). The results were validated using RT-qPCR by analysing 9 genes per experiment. The GeneSpring GX 11 analysis of the normalized data showed no change in the global gene expression profile of the oocytes after the HP treatment; however, in the 4-cell embryo study, the analysis identified 676 significantly changed genes, confirmed by the similar expression of 8 genes by RT-qPCR. To characterise the affected pathways among these genes, we used the functional annotation tools of DAVID Bioinformatics Resources (Huang et al. 2009 Nat. Protoc. 4, 44–57). The cluster with the most outstanding gene ontology terms contained the ribosome, structural constituent of ribosome, ribonucleoprotein complex, translation and structural molecule activity (P < 0.001). Among the 2 directly ribosome-related categories, there were 35 and 40 down-regulated genes along with only 2-2 up-regulated genes. These results showed that principally the protein synthesis machinery is affected by the HP stress, with a robust down-regulation. In conclusion, this study suggests that ribosomal processes play a central role in the HP-treated and fertilized oocytes during pre-implantation development. Stress effects in the transcriptional processes are visible after the embryonic genome activation showing down-regulation in the most energy-consuming processes. How these processes support higher blastocyst rate, cell number and birth rate needs further elucidation. Supported by OM-00069/2008, EGG_Care; EU FP7 (EpiHealth FP7-HEALTH-2011-278418).

Placental-specific microRNA in maternal circulation – identification of appropriate pregnancy-associated microRNAs with diagnostic potential

The goal of this study was to identify placental specific microRNAs present in maternal plasma that differentiate between women with normal pregnancies and nonpregnant individuals. The selection of appropriate pregnancy-associated microRNAs with diagnostic potential was based on the following criteria: (1) detection rate of 100% in full-term placentas, (2) detection rate of ≥ 67% in maternal plasma throughout gestation (at least four positive wells out of six tested wells) and (3) not detectable in whole peripheral blood and plasma samples of nonpregnant individuals. Initially, we tested microRNAs (miR-34c, miR-372, miR-135b and miR-518b), which had been previously identified as pregnancy-associated microRNAs. Additionally, we selected 16 other highly specific placental microRNAs (miR-512-5p, miR-515-5p, miR-224, miR-516-5p, miR-517*, miR-136, miR-518f*, miR-519a, miR-519d, miR-519e, miR-520a*, miR-520h, miR-524-5p, miR-525, miR-526a, and miR-526b) from the miRNAMap database. Seven microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) were identified as new pregnancy associated microRNAs with diagnostic potential. Their levels in maternal plasma during the 36th week of gestation corresponded to 45.0-427.0 pg of total RNA (enriched for small RNAs) per milliliter of maternal plasma.

Sensitive and Specific Universal RT-qPCR Profiling of miRNAs

RATIONALE: MicroRNAs (miRNAs) bind to target mRNAs and regulate their expression in a dose-dependent manner. The amount of specific miRNA in the cell is critical for the regulation process. Their expression pattern can be used as signatures of the developmental stage of cells and tissues, and as a biomarker for diagnosis and prognosis of disease, and miRNAs may be useful as therapeutic agents. Accurate quantitation of miRNAs is essential to their study. However, a highly specific and sensitive but simple method for quantitating miRNAs is still sought. METHODS: The method combines a universal hydrolysis probe with a universal reverse trasncription primer (UPR), which requires only one RT reaction and one hydrolysis probe for detection of all miRNAs. A computer program has been developed to design the probes and primers for the RT-qPCR assay. RESULTS: The method can detect and quantify miRNAs in as little as 1 pg of total RNA, and genomic DNA and mRNAs present in total RNA produce negligible or undetectable signals. Simple extraction methods such as Trizol (Invitrogen, CA, USA) without DNase treatment and small RNA fractionation is sufficient to produce total RNA for this assay. CONCLUSION: This approach affords a highly specific, sensitive, economical and convenient system to profile the expression of all known miRNAs.

Physicochemical properties of double-stranded RNA used to discover a reo-like virus from blue crab Callinectes sapidus

Mortality among blue crab Callinectes sapidus in soft shell production facilities is typically 25% or greater. The harvest, handling, and husbandry practices of soft shell crab production have the potential to spread or exacerbate infectious crab diseases. To investigate the possible role of viruses in soft shell crab mortalities, we took advantage of the physicochemical properties of double-stranded RNA (dsRNA) to isolate a putative virus genome. Further characterization confirmed the presence of a reo-like virus that possesses 12 dsRNA genome segments. The virus was present in >50% of dead or dying soft shell crabs, but fewer than 5% of healthy hard crabs. Injection of the virus caused mortality and resulted in the appearance of viral RNA and virus inclusions in hemocytes. The genome of the virus was partially sequenced and the information used to develop a reverse transcription polymerase chain reaction (RT-PCR) assay that is able to detect the virus genome in as little as 7.5 pg of total RNA. The molecular tools developed during this study will allow us to quantify prevalence of the blue crab reo-like virus in captive (soft shell facilities, aquaculture operations) and wild populations and facilitate understanding of the role this virus has in blue crab life history.

Circulating tyrosinase mRNA detection in a preclinical xenograft mouse melanoma model and in advanced melanoma patients

22204 Background: Although it is still under debate, molecular detection of circulating Tyrosinase is believed to be a good prognostic marker for melanoma progression. Methods: We have designed high-affinity primers to amplify the human Tyrosinase mRNA sequence to test the value of molecular detection of circulating melanoma cells by reverse transcription-PCR. The optimization process included in vitro settings and in vivo studies in a xenograft mouse model. Results: We were able to detect Tyrosinase expression with as few as 40 pg and 1.5 pg of total RNA from SKmel human melanoma cells, using PCR and nested PCR, respectively. Human Tyrosinase expression was found in the blood of nude mice bearing s.c. SKmel tumors, and expression bands were stronger after manipulation of the tumor mass. In this melanoma model, Tyrosinase expression declined in blood 6 hours after a direct intravenous injection of SKmel cells. A preliminary study in human blood samples demonstrated a baseline positive Tyrosinase determination in 64% (16/25) of advanced melanoma patients using the RT-PCR nested assay. Baseline Tyrosinase expression was significantly associated with disease progression after 12 months, and sequential determination during follow up of the remaining disease-free patients showed a progressive increase of negative results. Conclusions: We have developed sequential in vitro and in vivo preclinical validations to improve the optimization of the detection system. Our data indicate that molecular detection of circulating Tyrosinase mRNA in advanced melanoma patients could be considered a prognostic parameter. No significant financial relationships to disclose.

Real time TaqMan RT-PCR assay for the detection of Cucumber green mottle mosaic virus

A real time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of Cucumber green mottle mosaic virus (CGMMV). The method was designed to use a duo-primer system with a TaqMan probe targeting the conserved sequence in 3′ noncoding region (NCR) of CGMMV to detect isolates of this virus collected in China. The sensitivity of the real time RT-PCR assay was 0.13 pg of total RNA or 50 molecules of RNA transcripts. This level of sensitivity indicated that the one step real time RT-PCR developed in the present study could be used for routine testing assays. The real time RT-PCR method could assist in the implementation of quarantine measures for prevention and control of the disease caused by CGMMV.

An Improved Method of Reverse Transcription and Polymerase Chain Reaction (RT-PCR) to Efficiently Detect Potyvirus, Sweet potato feathery mottle virus (SPFMV) RNA in Sweet Potato

To improve the detection method for SPFMV in sweet potato leaves, we compared three RNA extraction methods that utilized either guanidine thiocyanate-phenol (ISOGEN™), SDS-phenol or a CF-11 cellulose column. The first method gave the highest yield of total RNA (39.0 μg from 0.1 g leaf sample) with the largest potential to synthesize cDNA by reverse transcription with the shortest extraction time. We then compared three combinations of polymerases for RT-PCR. AMV reverse transcriptase followed by PCR with rTaq DNA polymerase gave the most sensitive detection, in which the amplified DNA band was detected in a sample containing 0.2 pg of total RNA.

Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans

Studies have shown evidence of production of nitric oxide (NO) in adipose tissue, as well as inhibition of lipolysis by NO. We have analyzed nitric oxide synthase (NOS) expression in subcutaneous adipose tissue from 13 nonobese and 18 obese male subjects. Using a competitive reverse transcription polymerase chain reaction method, endothelial (eNOS) and inducible (iNOS), but not neuronal (nNOS), nitric oxide synthase mRNA expression was detected in isolated fat cells and pieces of adipose tissue. Tissue mRNA levels for eNOS were 3,814 +/- 825 and 5,956 +/- 476 amol/mg RNA (P = 0.043), and for iNOS 306 +/- 38 and 332 +/- 48 amol/mg RNA, for nonobese and obese individuals, respectively. Western blotting revealed similar eNOS protein levels in isolated fat cells and adipose tissue pieces. Protein levels for eNOS in nonobese and obese individuals, respectively, were (in optical density [OD] units per mm(2) per 100 microgram of total protein) 0.11 +/- 0.08 and 2.80 +/- 1.30 (P = 0.043). iNOS protein was detectable, but not measurable, at low levels in a subset of obese patients (3 of 10). iNOS protein levels could not be detected in nonobese individuals. Hormone-sensitive lipase (HSL), the key regulating enzyme in lipolysis, is reduced in obesity. The expression of HSL protein in subcutaneous adipose tissue was studied in the same subset of patients; in agreement with previous results, HSL levels were reduced in obese subjects: 4.64 +/- 1.10 and 1.27 +/- 0.35 (P = 0.012) in nonobese and obese subjects, respectively. In conclusion, this study shows that eNOS and iNOS, but not nNOS, are present in human subcutaneous adipose tissue. Gene expression and protein levels of eNOS are increased, whereas HSL protein levels are decreased in obesity. It is speculated that increased NO production, preferably by eNOS, and decreased HSL levels may cause decreased subcutaneous adipose tissue lipolysis in obesity. synthases in subcutaneous adipose tissue of nonobese and obese humans.