Strategies for microarray analysis of limiting amounts of RNA.

One of the critical limitations of current microarray technologies for use in expression analyses is the relatively large amount of input RNA required to generate labelled cDNA populations for array analysis. In situations where RNA is limiting, the options for expression profiling are to increase cDNA labelling and hybridisation efficiency, or to use an amplification strategy to generate enough RNA/cDNA for use with a standard labelling method. Sample amplification approaches must preserve the representation of the relative abundances of the different RNAs within the starting population and must also be highly reproducible. This review evaluates current signal and sample amplification technologies, including those that can be used to generate labelled cDNA populations for array analysis from as little as a single cell.     

Somaclonal variation does not preclude the use of rice transformants for genetic screening

Rice (Oryza sativa) is one of the world's most important crops. Rice researchers make extensive use of insertional mutants for the study of gene function. Approximately half a million flanking sequence tags from rice insertional mutant libraries are publicly available. However, the relationship between genotype and phenotype is very weak. Transgenic plant assays have been used frequently for complementation, overexpression or antisense analysis, but sequence changes caused by callus growth, Agrobacterium incubation medium, virulence genes, transformation and selection conditions are unknown. We used high‐throughput sequencing of DNA from rice lines derived from Tainung 67 to analyze non‐transformed and transgenic rice plants for mutations caused by these parameters. For comparison, we also analyzed sequence changes for two additional rice varieties and four T‐DNA tagged transformants from the Taiwan Rice Insertional Mutant resource. We identified single‐nucleotide polymorphisms, small indels, large deletions, chromosome doubling and chromosome translocations in these lines. Using standard rice regeneration/transformation procedures, the mutation rates of regenerants and transformants were relatively low, with no significant differences among eight tested treatments in the Tainung 67 background and in the cultivars Taikeng 9 and IR64. Thus, we could not conclusively detect sequence changes resulting from Agrobacterium‐mediated transformation in addition to those caused by tissue culture‐induced somaclonal variation. However, the mutation frequencies within the two publically available tagged mutant populations, including TRIM transformants or Tos17 lines, were about 10‐fold higher than the frequency of standard transformants, probably because mass production of embryogenic calli and longer callus growth periods were required to generate these large libraries.     

An antibody-based microarray assay for small RNA detection

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 µl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.     

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.     

Postdifferential display: parallel processing of candidates using small amounts of RNA

The necessity of screening differentially expressed candidate genes has imposed a limit on the application of differential display to large-scale analysis of gene expression patterns. Screening candidates has indeed proven a burden because traditional screening methods require the purification of large amounts of RNA. In this article we describe an assay that allows the screening of 240 candidate genes with only 5 microg of total RNA. This assay consists of using cDNA probes synthesized from amplified RNA in differential screening and can be performed in a 96-well plate format.     

Moderate summer heat stress does not modify immunological parameters of Holstein dairy cows

The study was undertaken during spring and summer months in a territory representative of the Mediterranean climate to assess the effects of season on some immunological parameters of dairy cows. Twenty Holstein cows were used. Eleven of those cows gave birth during spring; the remaining nine cows gave birth in summer. The two groups of cows were homogeneous for parity. Values of air temperatures and relative humidity were recorded both during spring and summer, and were utilized to calculate the temperature humidity index (THI). One week before the expected calving, rectal temperatures and respiratory rates of the cows were recorded (1500 hours), and cell-mediated immunity was assessed by measuring the proliferation of mitogen-stimulated peripheral blood mononuclear cells (PBMC). Within 3 h of calving, one colostrum sample was taken from each cow and analysed to determine content of immunoglobulin (Ig) G1, IgG2, IgM and IgA. At 48 h after birth, passive immunization of the calves was assessed by measuring total serum IgG. During summer, daytime (0900-2000 hours) THI values were above the upper critical value of 72 [75.2, (SD 2.6)] indicating conditions that could represent moderate heat stress. That THI values were able to predict heat stress was confirmed by the values of rectal temperatures and respiratory rates, which were higher (P < 0.05 and P < 0.001 respectively) during summer. Proliferation of PBMC, the colostral concentration of Ig fractions and serum levels of IgG in their respective off-spring did not differ between spring and summer cows. Results indicated that moderate heat stress due to the hot Mediterranean summer does not modify cell-mediated immunity, the protective value of colostrum and passive immunization of the offspring in dairy cows.     

Isolation of functional RNA from small amounts of different grape and apple tissues

An efficient, simple, and small-scale procedure for isolating functional ribonucleic acid (RNA) was successfully applied to many different tissues of grape and apple. These woody plants are rich in polyphenolic compounds and polysaccharides that could impair the RNA extraction. The method chosen is based on the use of hot borate buffer at alkaline pH supplemented with several adjuvants and followed by selective precipitations. Starting with only 0.4 g of fresh tissue and working with small tubes (2 mL), we were able to obtain good yields of high-quality RNA suitable for further applications. The procedure can be proposed for many applications, and it is particularly highly recommended when isolating RNA from a large number of samples.     

High fidelity does not preclude colonization: range expansion of molting Black Brant on the Arctic coast of Alaska

High rates of site fidelity have been assumed to infer static distributions of molting geese in some cases. To test this assumption, we examined movements of individually marked birds to understand the underlying mechanisms of range expansion of molting Black Brant (Branta bernicla nigricans) on the Arctic Coastal Plain (ACP) of Alaska. The Teshekpuk Lake Special Area (TLSA) on the ACP was created to protect the primary molting area of Brant. When established in 1977, the TLSA was thought to include most, if not all, wetlands used by molting Brant on the ACP. From 2010 to 2013, we surveyed areas outside the TLSA and counted an average of 9800 Brant per year, representing 29–37% of all molting Brant counted on the ACP. We captured and banded molting Brant in 2011 and 2012 both within the TLSA and outside the TLSA at the Piasuk River Delta and Cape Simpson to assess movements of birds among areas across years. Estimates of movement rates out of the TLSA exceeded those into the TLSA, demonstrating overall directional dispersal. We found differences in sex and age ratios and proportions of adult females with brood patches, but no differences in mass dynamics for birds captured within and outside the TLSA. Overall fidelity rates to specific lakes (0.81, range = 0.49–0.92) were unchanged from comparable estimates obtained in the early 1990s. We conclude that Brant are dispersing from the TLSA into new molting areas while simultaneously redistributing within the TLSA, likely as a consequence of changes in relative habitat quality. Shifts in distribution resulted from colonization of new areas by young birds as well as low levels of directional dispersal of birds that previously molted in the TLSA. Based on combined counts, the overall number of molting Brant across the ACP has increased substantially.     

Moderate severity heart failure does not involve a downregulation of myocardial fatty acid oxidation

Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of free fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,10-(3)H]oleate, [U-(14)C]glucose, and [1-(13)C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity for malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomenon.     

A small molecule microarray platform to select RNA internal loop-ligand interactions.

Herein, we report the development of a microarray platform to select RNA motif-ligand interactions that allows simultaneous screening of both RNA and chemical space. We used this platform to identify the RNA internal loops that bind 6'- N-5-hexynoate kanamycin A ( 1). Selected internal loops that bind 1 were studied in detail and commonly display an adenine across from a cytosine independent of the size of the loop. Additional preferences are also observed. For 3 x 3 nucleotide loops, there is a preference for purines, and for 2 x 2 nucleotide loops there is a preference for pyrimidines neighbored by an adenine across from a cytosine. This technique has several advantageous features for selecting RNA motif-ligand interactions: (1) higher affinity RNA motif-ligand interactions are identified by harvesting bound RNAs from lower ligand loadings; (2) bound RNAs are harvested from the array via gel extraction, mitigating kinetic biases in selections; and (3) multiple selections are completed on a single array surface. To further demonstrate that multiple selections can be completed in parallel on the same array surface, we selected the RNA internal loops from a 4096-member RNA internal loop library that bound a four-member aminoglycoside library. These experiments probed 16,384 (4 aminoglycoside x 4096-member RNA library) interactions in a single experiment. These studies allow for parallel screening of both chemical and RNA space to improve our understanding of RNA-ligand interactions. This information may facilitate the rational and modular design of small molecules targeting RNA.     

Reanalysis and simulation suggest a phylogenetic microarray does not accurately profile microbial communities

The second generation (G2) PhyloChip is designed to detect over 8700 bacteria and archaeal and has been used over 50 publications and conference presentations. Many of those publications reveal that the PhyloChip measures of species richness greatly exceed statistical estimates of richness based on other methods. An examination of probes downloaded from Greengenes suggested that the system may have the potential to distort the observed community structure. This may be due to the sharing of probes by taxa; more than 21% of the taxa in that downloaded data have no unique probes. In-silico simulations using these data showed that a population of 64 taxa representing a typical anaerobic subterranean community returned 96 different taxa, including 15 families incorrectly called present and 19 families incorrectly called absent. A study of nasal and oropharyngeal microbial communities by Lemon et al (2010) found some 1325 taxa using the G2 PhyloChip, however, about 950 of these taxa have, in the downloaded data, no unique probes and cannot be definitively called present. Finally, data from Brodie et al (2007), when re-examined, indicate that the abundance of the majority of detected taxa, are highly correlated with one another, suggesting that many probe sets do not act independently. Based on our analyses of downloaded data, we conclude that outputs from the G2 PhyloChip should be treated with some caution, and that the presence of taxa represented solely by non-unique probes be independently verified.