Extraction of high-quality RNA from pancreatic tissues for gene expression studies

Extracting RNA from pancreatic tissue is notoriously challenging because of the organ's high RNase content. Standard methods using TriPure or TRIzol classically yield RNA of sufficient quality for routine gene expression analysis but not for microarray or deep sequencing analysis. Here we developed a simple method to extract high-quality RNA from mouse pancreas. Our method uses an RNase inhibitor and combines different protocols using guanidium thiocyanate–phenol extraction. It enables reproducible isolation of RNA with an RNA integrity number around 9.     

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.     

A monophasic solution for isolation of RNA devoid of polymerase chain reaction-detectable genomic DNA contamination

Commercially available reagents and published protocols are widely used for RNA isolation. However, genomic DNA contamination in isolated RNA is a potential problem. Here we describe a simple, inexpensive method for eliminating genomic DNA contamination beyond the level of PCR-based detection through reduction of the guanidine thiocyanate concentration (1.5 M) in a single monophasic solution based on Chomczynski–Sacchi reagents. The new method can be used to isolate small and large RNA species of high quality and can be completed within an hour.     

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle

We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at −80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.     

Self assembly of poly(o-methoxy aniline) with RNA and RNA/DNA hybrids: Physical properties and conformational change of poly(o-methoxy aniline)

Biomolecular hybrids of a conducting polymer [poly(o-methoxy aniline) (POMA)] and RNA are prepared at the three different compositions by mixing aqueous solutions of diethyl, 2-hydroxy ethyl, ammonium salt of RNA (type IX from Torula Yeast) and POMA (ES, emeraldine salt; doping level [Cl]/[N] = 0.52). A slow increase of pH up to 30 h of aging occurs in the mixture till it levels up. The TEM micrographs indicate a fibrillar network structure in all the hybrid compositions (POMA: RNA = 1:3, 1:1, 3:1, by weight). In the complexes three types of supramolecular interactions, viz. (i) electrostatic, (ii) H-bonding and (iii) π–π interactions, are evident from the FTIR spectroscopy. The CD spectra indicate a small distortion of A-RNA conformation towards its B form during the hybrid formation. Time and temperature dependent UV–vis spectral studies indicate a slow red shift of the π-band to polaron band transition peak (λ_max) for the uncoiling of the POMA (P) chain on the RNA (R) surface. The repulsive interaction between the radical cations of POMA (ES) absorbed on the RNA surface is attributed to the conformational change causing the uncoiling of POMA chain. UV–vis spectral study indicates that the uncoiling and attachment of POMA on RNA surface is much faster than that on DNA (D). In POMA–RNA–DNA (PRD) hybrid solutions slower red shift of λ_max indicates more disordered array of the phosphate groups than that in PR and PD systems. The conductivity values of the PR hybrids (10^− 6 S/cm^− 1) are three orders higher than that of RNA, rendering the PR hybrids to be useful for fabricating good biosensors. In the PRD hybrids conductivity decreases by two orders than those of PR and PD hybrids suggesting a disorder arrangement of POMA chains in the PRD hybrids. The I–V characteristic curves of the PR and PRD hybrids indicate a semiconducting nature of the hybrids.     

Crystal structure of the single-stranded RNA binding protein HutP from Geobacillus thermodenitrificans

RNA binding proteins control gene expression by the attenuation/antitermination mechanism. HutP is an RNA binding antitermination protein. It regulates the expression of hut operon when it binds with RNA by modulating the secondary structure of single-stranded hut mRNA. HutP necessitates the presence of l-histidine and divalent metal ion to bind with RNA. Herein, we report the crystal structures of ternary complex (HutP–l-histidine–Mg²⁺) and EDTA (0.5 M) treated ternary complex (HutP–l-histidine–Mg²⁺), solved at 1.9 Å and 2.5 Å resolutions, respectively, from Geobacillus thermodenitrificans. The addition of 0.5 M EDTA does not affect the overall metal-ion mediated ternary complex structure and however, the metal ions at the non-specific binding sites are chelated, as evidenced from the results of structural features.     

何首乌总RNA提取方法的比较及改进

目的：探讨不同提取方法对提取何首乌总RNA的质量影响,寻找适于何首乌成熟叶组织RNA的提取方法。方法：以何首乌成熟叶组织为材料,采用SDS／酸酚法、常规CTAB法及改良的TRIzol试剂法分别进行实验,并对所提取RNA的质量进行验证。结果：采用3种方法都能提取出RNA,但质量差异较大。其中改良的TRIzol试剂法能有效抑制次生物质的影响,提取的RNA产量可达70-110μg／g,纯度高于其他2种方法,D260nm／D280nm值为1．85～1．97。结论：改良的TRIzol试剂法操作简便,提取的RNA完整性和纯度较高,可以满足下一步实验的要求。     

Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm

Different cholesterol conjugates-loaded-cyclodextrin was added to bull sperm to improve sperm quality after freezing. Ejaculates from four bulls were diluted to 120 million cells/ml in Tris (T) diluent and then sub-divided into 10 treatments as follow: no additive (control); 1.5 mg CLC (positive control); 0.75 mg or 1.5 mg of cyclodextrin pre-loaded with cholesterol conjugated to heptanoate, palmitate, pelargonate or stearate, and incubated for 15 min at 22 °C. The samples were then diluted 1:1 with 20% egg yolk (EY) in T diluent and cooled to 5 °C over a 2 h. Upon reaching 5 °C, the samples were diluted 1:1 with T diluent containing 10% EY + 16% glycerol, and allowed to equilibrate for 15 min, and packaged into 0.5 ml straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the sperm motility, viability and number sperm binding to perivitelline membrane were determined. The ability of bull sperm to bind to the oocyte membranes was conducted using the chicken egg perivitelline membrane (CEPM) as described by Barbato et al. [G.F. Barbato, P.G. Cramer, R.H. Hammerstedt, A practical in vitro sperm-egg binding assay that detects subfertile males. Biol. Reprod. 58 (1998) 686-699] and modified by Amorim et al. [E. Amorim, J.K. Graham, B. Spizziri, M. Meyers, L. Amorim, C. Torres, The effect of adding cholesteryl-heptanoate, -palmitate, -pelargonate, or -stearate loaded cyclodextrin on bull sperm cryosurvival, in: Proceeding 40th Annual Meeting of the Society for the Study of Reproduction (SSR), July, San Antonio, TX, EUA, 2007], where these authors did not observe difference between bovine zona pellucide and CEPM. Higher percentages of motile and viable sperm were maintained after thawing from bull sperm treated with CLC and pelargonate compared to all other treatments (P < 0.05). Control samples and sperm treated with heptanoate, palmitate, or stearate loaded cyclodextrin exhibited similar motility percentages and viable sperm after freezing (P > 0.05). The percentage of motile sperm and number sperm binding to chicken egg perivitelline membrane was higher for CLC and pelargonate compared to control (P < 0.05). Therefore, adding either cholesterol or pelargonate to bull sperm membranes improved cell cryosurvival, whereas treatments with cyclodextrins pre-loaded with other cholesterol-like molecules did not.     

条斑紫菜丝状体总RNA提取方法比较

目的:为了获得质量较高的条斑紫菜丝状体总RNA,对几种常用提取方法进行研究。方法:以条斑紫菜自由丝状体为材料,比较了用异硫氰酸胍法、CTAB法、SDS/酚法、TRIzol法、RNAplant法提取的RNA的质量和纯度。结果:异硫氰酸胍法提取RNA的成本低,但纯度不高;CTAB法产率较小,且不能完全去除多糖或蛋白质;SDS/酚法未能获得完整的RNA;TRIzol法未能见到5SrRNA条带,且带有杂带;而RNAplant法提取RNA的质量好、纯度高、提取效率高,其D260nm/D280nm值为1.836,经逆转录得到的双链cDNA扩增产物长度在200bp以上。结论:实验结果表明RNAplant法更适于条斑紫菜丝状体总RNA的提取。     

The complete mitochondrial genomes of three grasshoppers, Asiotmethis zacharjini, Filchnerella helanshanensis and Pseudotmethis rubimarginis (Orthoptera: Pamphagidae)

The complete mitogenomes of Asiotmethis zacharjini, Filchnerella helanshanensis and Pseudotmethis rubimarginis are 15,660 bp, 15,657 bp and 15,661 bp in size, respectively. All three mitogenomes contain a standard set of 13 protein - coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and an A + T-rich region in the same order as those of the other analysed caeliferan species, including the rearrangement of trnAsp and trnLys. The putative initiation codon for the cox1 gene in the three species is CCG. The long polythymine stretch (T-stretch) in the A + T-rich region of the three species is not adjacent to the trnIle but inside the stem–loop sequence in the majority strand. The mitogenomes of F. helanshanensis and P. rubimarginis have higher overall similarities. The characterization of the three mitogenomes will enrich our knowledge on the Pamphagidae mitogenome. The phylogenetic analyses indicated that within the Caelifera, Pyrgomorphoidea is a sister group to Acridoidea. The species from the Pamphagidae form a monophyletic group, as is the case for Acrididae. Furthermore, the two families cluster as sister groups, supporting the monophyly of Acridoidea. The relationships among eight acridid subfamilies were (Cyrtacanthacridinae + (Calliptaminae + (Catantopinae + (Oxyinae + (Melanopline + (Acridinae + (Oedipodinae + Gomphocerinae))))))).     

仿刺参体壁总RNA提取方法的建立

目的:通过对TRIzol一步法进行改进,建立一种从富含胶原蛋白、多糖及色素的仿刺参体壁提取总RNA的有效方法。方法:样品在液氮中研磨并用TRIzol匀浆后再进行抽提;对TRIzol一步法提取的总RNA进行DNaseⅠ消化和酚氯仿抽提,用2.5mol/L的醋酸钾沉淀,并加入适量糖原(10mg/mL)与RNA共沉淀。结果:琼脂糖凝胶电泳和紫外分光光度法以及RT-PCR检测结果表明,改进的方法能够有效去除基因组DNA、蛋白、多糖及色素的污染,RNA的产率提高。结论:制备的总RNA纯度高,完整性好,能够满足mRNA差异显示RT-PCR等分子生物学研究的要求,是一种提取仿刺参体壁及其他富含黏多糖、胶原蛋白和色素的动物组织总RNA的有效方法。     

Superovulation and embryo transfer in Holstein cattle using sexed sperm

The use of sexed bull sperm in multiple ovulation and embryo transfer (MOET) programs for Holsteins was evaluated for (1) heifers housed at a commercial embryo transfer (ET) facility (Experiments 1 and 2), and (2) heifers and cows on dairy farms (Experiment 3). In Experiment 1, superstimulated heifers were inseminated with 5 × 10⁶ sexed (X-sorted; n = 5) or unsexed (n = 5) frozen-thawed sperm from one bull at 12 and 24 h after estrus detection. No difference was observed in the rates of transferable embryos (53.4% vs 68.1%), degenerate embryos (24.8% vs 26.6%) and unfertilized ova (21.8% vs 5.3%) between sexed and unsexed sperm, respectively, except for the percent of female transferable embryos diagnosed by embryo sexing (100% vs 49.3%, P < 0.0001). In Experiment 2, donors were inseminated twice with 5 × 10⁶ sexed unfrozen sperm (n = 10) or sexed frozen-thawed sperm (n = 9). Embryo production rates for both treatments were similar to that observed on a commercial ET facility using unsexed sperm. Pregnancy rates for frozen-thawed embryos were similar for sexed and unsexed sperm (70.4% vs 72.4%, respectively). In Experiment 3, 99 flushes were conducted using sexed frozen-thawed sperm from nine bulls but an overall statistical analysis was not completed because the use of bulls was not balanced. However, for one bull with balanced usage, the rate of transferable embryos was higher in heifers than in cows (P < 0.05) inseminated twice with 5 × 10⁶ sperm/dose (10 × 10⁶ total). We concluded that the use of sexed frozen-thawed sperm (≥90% X-sperm biased and 10 × 10⁶ total sperm) may be economically viable for commercial MOET programs in Holstein heifers.     

Optimization of total RNA isolation from Cochlodinium polykrikoides

Dinoflagellates are eukaryotic microorganisms that represent a group of single-celled algae composed of a highly diversified phylum that is well known for displaying an amazing range of ecological adaptation. Cochlodinium polykrikoides is an unarmored dinoflagellate species belonging to the Family Gymnodiniaceae. It is one of the causative agents for harmful algal blooms occurring in the west coast of Sabah, which result in massive economic damages to the aquaculture and mariculture industries. The limited availability of information pertaining to the genetics of C. polykrikoides raises the need for an in-depth study into the expression mechanisms and regulation of the genes of this microorganism. However, isolation of good quality and intact RNA samples from C. polykrikoides seems to be quite a challenge for molecular biologists. Rapid degradation of freshly isolated RNA samples is a bottleneck for researchers to conduct studies on the gene expression and regulation patterns, which requires intact and high-quality RNA samples. In this study, an attempt to establish the best protocol for isolation of good quality and intact RNA from C. polykrikoides was carried out by testing four different total RNA isolation protocols. We conclude that the RNA isolation protocol using the TRIzol reagent produced the best results among the methods tried. The information from this study will allow further research pertaining to the molecular aspects of C. polykrikoides to be conducted with much ease in the future.     

A model to estimate growth in young-of-the-year tautog, Tautoga onitis, based on RNA/DNA ratio and seawater temperature

This study presents an indirect method for estimating growth rates of young-of-the-year (YOY) tautog, Tautoga onitis, based on laboratory calibration experiments and nucleic acid-based indices. Field-collected tautog were held in the laboratory at 3 temperatures over a 17-day period. Four feeding levels were used to produce a range of growth rates. An ultraviolet absorption assay was used to measure nucleic acid concentrations in white muscle tissue. The strength of the relationship between growth rate and three nucleic acid-based parameters (RNA concentration, DNA concentration, RNA/DNA ratio (R/D)) was tested. Correlation results indicated a significant positive relationship between R/D and weight-based instantaneous growth rate (G) (r = 0.68; p < 0.001). Both R/D (r = − 0.55; p < 0.006) and RNA (r = − 0.56; p < 0.005) were highly negatively correlated with temperature (T). Multiple linear regression showed that R/D and temperature explained 61% of the variability in growth, resulting in the model G = 0.01285(R/D) + 0.00057(T) − 0.03205 (p < 0.0001). This R/D-temperature model can be used to evaluate recent growth rates in YOY tautog under field conditions and has applications for aquaculture when comparing growth rates of fish held under different culture conditions.