Reduced Graphene Oxide-Based Solid-Phase Extraction for the Enrichment and Detection of microRNA

MicroRNAs (miRNAs) are endogenous molecules with regulatory functions. The purification and enrichment of miRNA are essential for its precise and sensitive detection. miRNA isolated using commercial kits contains abundant interfering RNAs, and the concentration of miRNA may not be adequate for detection. Herein, we prepared a reduced graphene oxide (rGO)-based magnetic solid-phase extraction material for the enrichment and ultrasensitive detection of miRNA from intricate nucleic acid solutions. <i>In situ</i> reverse transcription (RT) was developed as the most efficient approach to desorb miRNA from rGO among the methods that are compatible for the subsequent amplification reported thus far. Additionally, rolling circle amplification and qPCR were used to detect let-7a with a decrease of the limit of detection by 24.7- and 31.3-fold, respectively. This material was also successfully used to extract and detect miRNA from total RNA isolated from human plasma. Our results show that the material prepared in this study has the potential for cancer biopsy in clinics and the discovery of new miRNAs in scientific research

Biological-Profiling-Based Systematic Analysis of Rhizoma Coptidis from Different Growing Regions and Its Anticholesterol Biosynthesis Activity on HepG2 Cells

Rhizoma Coptidis is a widely cultivated traditional Chinese herb. Although the chemical profiles of Rhizoma Coptidis have been established previously, the biological profiling of Rhizoma Coptidis has not been conducted yet. In this study, we collected Rhizoma Coptidis varieties from four distinct growing regions and performed genome-wide biological response fingerprinting (BioReF) on HepG2 cells using a gene expression array. Similar biological pathways were affected by extracts of all four Rhizoma Coptidis varieties but not by their analogue, Mahoniae Caulis. Among these pathways, the terpenoid backbone biosynthesis pathway was highly enriched, and six genes in the mevalonate (MVA) pathway were all down-regulated. However, the expression, maturation, as well as the specific DNA binding capacity of their coordinate transcription factor, sterol response element binding protein 2 (SREBP2), was not affected by Rhizoma Coptidis extract (RCE) or its typical active alkaloid berberine. Cellular cholesterol content tests further verified the cholesterol-lowering function of RCE in vitro, which supplements evidence for the use of Rhizoma Coptidis in hyperlipidemia treatment. This is the first described example of evaluating the quality of Rhizoma Coptidis with BioReF and a good demonstration of using BioReF to uncover the mechanisms of herbs at a systematic level

Microarray design.

<p>(A) Copy of the microarray; (B) the microarray layout. QC: chemical control; PC: external control; BC: blank control; NC: negative control; IC: internal control; MC: magnetic control; W: wild-type; M: mutant.</p

The principle for design of the amplification primers.

<p>Multiplex PCR is performed as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169219#pone.0169219.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169219#pone.0169219.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169219#pone.0169219.ref029" target="_blank">29</a>]. Asymmetric PCR procedure was used for generation of labeled single-stranded DNA in excess for hybridization and detection.</p

Mutations detected in the deaf individuals.

<p>Mutations detected in the deaf individuals.</p

The percentage of bacteria identified as definite (dark blue) and possible (light blue) causative agents in LRTI patients (for the full bacterial names see the legend of

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038743#pone-0038743-g002" target="_blank"><b>Figure 2</b></a><b>).</b> The patients are classified as: children, ≤14 yr; middle-aged adult, >14 yr but <70 yr; aged, ≥70 yr; AB, acute bronchitis; CAP, community acquired pneumonia; AECOPD, acute exacerbation of COPD; and AEBX, acute exacerbation of bronchiectasis.</p

Data Summary of qLAMP and culture assays.

<p>Note: The Abbreviations are: Ab, <i>A. baumannii</i>; Ec, <i>E. coli</i>; Hi, <i>H. influenzae</i>; Kp, <i>K. pneumoniae</i>; Pa, <i>P. aeruginosa</i>; Sa, <i>S. aureus</i>; Sm, <i>S. maltophilia</i>; and Sp, <i>S. pneumonia</i>.</p>*<p>indicates the number of patients whose positive culture was confirmed by one of the 4 culture-based tests.</p>**<p>indicate confirmation rate of the positive cultures by one of the 4 culture-based tests.</p>***<p>indicate the bacterial mortality due to refrigeration, storage, and transportation.</p

Relatedness between qLAMP and culture assays and cutoffs in different subgroups.

<p>Note: We only used the data from the three-time culture to test the consistency between qLAMP and culture assays. N and BX stand for not found and bronchiectasis, respectively.</p>*<p>In the piecewise linear regression of <i>S. pneumoniae</i> for child patients, all the detected titers were PCs (pathogen candidates), and no breakpoint was found. Therefore, the lowest titer detected in this subgroup is deemed as the upper cutoff.</p

qLAMP and culture result from LRTI patients.

<p>(A) The positive rates (the right vertical axis) of one-time culture (brown bar), three-time culture (blue bar), and quantitative LAMP (yellow bar) for the eight species in the panel (from the left: Ab, <i>A. baumannii</i>; <i>Ec, E. coli;</i> Hi, <i>H. influenzae;</i> Kp, <i>K. pneumoniae;</i> Pa, <i>P. aeruginosa;</i> Sa, <i>S. aureus</i>; Sm, <i>S. maltophilia;</i> and Sp, <i>S. pneumoniae</i>) detected from the number of patients (the left vertical axis). (B) The number of patients (the left vertical axis) who were tested positive for at least one bacterium in one-time culture, three-time culture, and qLAMP. Each bar is the sum of patient with single (blue bar) and multiple (yellow bar) species detected.</p

Examples of <i>S. pneumonia</i> showing the relationship between qLAMP and culture results (logistic regression) and cutoff determination based on competitive relationship (piece-wise linear regression).

<p>The horizontal axis displays the bacterial natural logarithmic titer in sputum sample. (A) Logistic regression curve (green line). Solid circles indicate patients; they are placed at the top of the chart when being test as positive and at the bottom of the chart when being tested as negative in the culture assays. The height and width of the bars display the frequency and the number of patients being tested positive in cultures, respectively. (B) Piecewise linear regression (black lines) of <i>S. pneumonia</i> in COPD patients. Open circles indicate patients; they are placed at the top of the chart when being PC (Pathogen Candidate) and at the bottom of the chart when NOT being PC.</p