Sensitive, Low-Background-Signal miRNA Analysis via the Self-Priming-Initiated Color Reaction Loaded on a Rolling Circle Amplification Product

MicroRNAs (miRNAs) have been regarded as potential biomarkers in evaluating various diseases, such as pregnancy-induced hypertension and cancers. However, sensitive and reliable miRNA detection is still a challenge due to the low amplification efficiency and high background signal. Herein, we developed a colorimetric method for miRNA detection utilizing the self-priming-initiated color reaction loaded on a rolling circle amplification (RCA) product. In this method, a biotin-labeled RCA product is fixed on the surface of the streptavidin-coated wells, and the interfering components in samples are removed to avoid false reactions, thus reducing the background signals. Two signal amplification processes, including RCA and self-priming-initiated chain extension, endow the method with high sensitivity and a low limit of detection at the 10 fM level. In conclusion, our approach offers a promising perspective on sensitive and reliable miRNA detection and has the potential to be further utilized in biomedical research and early cancer detection

symbiont-mediated suppression of induced-defenses

symbiont-mediated suppression of induced-defense

Effect of <i>Tomato yellow leaf curl virus</i> infection on symbiont densities (as indicated by relative copy number of specific genes) in <i>B. tabaci</i> B.

<p>(A) <i>Portiera</i> density in 2-week-old males and females. (B) <i>Hamiltonella</i> density in 2-week-old males and females. (C) <i>Rickettsia</i> density in 2-week-old males and females. (D) <i>Rickettsia</i> density in 4-week-old males and females; V(–) (non-viruliferous), V(+) (viruliferous). Sample sizes are indicated above the bars. *indicates significant a significant (<i>p</i><0.001) difference between that treatment and the other treatments. Values are means±SE.</p

Fluorescence <i>in situ</i> hybridization of <i>B. tabaci</i> nymphs exposed to TYLCV.

<p>(A) Double FISH analysis of a non-viruliferous <i>B. tabaci</i> nymph with specific probes for <i>Portiera</i> (red) and <i>Rickettsia</i> (blue). (B) Double FISH analysis of a viruliferous <i>B. tabaci</i> nymph with specific probes for <i>Portiera</i> (red) and <i>Rickettsia</i> (blue). Bars = 100 µm. (C) Mean fluorescence intensity (MFI). Fluorescence was quantified from 20 viruliferous nymphs and 20 non-viruliferous nymphs by counting the pixels in the corresponding image fields in each group. Pixel intensity was then quantified and finally expressed as MFI. Different letters indicate significant differences between virus treatments (<i>P</i><0.05).</p

Whole-mount FISH analysis of the <i>B. tabaci</i> B nymphs used in this study.

<p>(A) Overlay of <i>Portiera</i> and <i>Rickettsia</i> on dark field. (B) Overlay of <i>Portiera</i> and <i>Hamiltonella</i> on dark field. The epifluorescent images were generated artificially by combining two relevant monochrome images obtained with the same exposure time in the microscope. Red: <i>Portiera</i>; Blue: <i>Rickettsia</i>. Green: <i>Hamiltonella</i>. Bars = 100 µm.</p

Population density of bacterial symbionts (as indicated by relative copy number of specific genes) during <i>B. tabaci</i> B development and as affected by <i>B. tabaci</i> B mating status.

<p>(A) <i>Portiera</i> density. (B) <i>Hamiltonella</i> density. Whitefly sexes are indistinguishable before the adult phase (earlier than day 20 after hatch). Sample sizes are indicated above the data symbols for each time point. *, **indicate significant differences between densities in virgin males and females at the indicated time point (<i>p</i><0.01, 0.001; respectively). (C) <i>Portiera</i> and <i>Hamiltonella</i> densities in 1-, 2-, 3-, and 4-week-old mated females. Values are means±SEM (n ≥10).</p

Symbiont density (as indicated by relative copy number of specific genes) in <i>B. tabaci</i> whiteflies through three successive host generations maintained under different temperature conditions.

<p>(A) <i>Portiera</i> density. (B) <i>Hamiltonella</i> density. (C) <i>Rickettsia</i> density. Values are means±SE (n ≥10). Different letters indicate significant differences among temperature treatments in the same generation (<i>P</i><0.05).</p

Critical Analysis of Multi-Omic Data from a Strain of Plutella xylostella Resistant to Bacillus thuringiensis Cry1Ac Toxin

Rapid evolution of resistance in crop pests to Bacillus thuringiensis (Bt) products threatens their widespread use, especially as pests appear to develop resistance through a range of different physiological adaptations. With such a diverse range of mechanisms reported, researchers have resorted to multi-omic approaches to understand the molecular basis of resistance. Such approaches generate a lot of data making it difficult to establish where causal links between physiological changes and resistance exist. In this study, a combination of RNA-Seq and iTRAQ was used with a strain of diamondback moth, Plutella xylostella (L.), whose resistance mechanism is well understood. While some of the causal molecular changes in the resistant strain were detected, other previously verified changes were not detected. We suggest that while multi-omic studies have use in validating a proposed resistance mechanism, they are of limited value in identifying such a mechanism in the first place

Statistical parsimony network of <i>Plutella xylostella</i> mt<i>COI</i> haplotypes.

<p>The red and blue circles represents shared and unique haplotypes, respectively. Haplotype names are beside the circles. The small circles indicate the presence of missing intermediates, while the connections are based on the set of plausible solutions with a 95% of parsimony probability.</p

Locations where <i>Plutella xylostella</i> populations were sampled in China.

<p>Arrows indicate possible migration routes based on shared haplotypes. The software Adobe Photoshop CS6, Micosoft PowerPoint 2013 and Micosoft Word 2013 were used to create and modify this map.</p