The Impact of Blood Sample Processing on Ribonucleic Acid (RNA) Sequencing.

In gene quantification and expression analysis, issues with sample selection and processing can be serious, as they can easily introduce irrelevant variables and lead to ambiguous results. This study aims to investigate the extent and mechanism of the impact of sample selection and processing on ribonucleic acid (RNA) sequencing. RNA from PBMCs and blood samples was investigated in this study. The integrity of this RNA was measured under different storage times. All the samples underwent high-throughput sequencing for comprehensive evaluation. The differentially expressed genes and their potential functions were analyzed after the samples were placed at room temperature for 0h, 4h and 8h, and different feature changes in these samples were also revealed. The sequencing results showed that the differences in gene expression were higher with an increased storage time, while the total number of genes detected did not change significantly. There were five genes showing gradient patterns over different storage times, all of which were protein-coding genes that had not been mentioned in previous studies. The effect of different storage times on seemingly the same samples was analyzed in this present study. This research, therefore, provides a theoretical basis for the long-term consideration of whether sample processing should be adequately addressed.

Agarose amplification based sequencing characterization cell-free RNA in preimplantation spent embryo medium.

BACKGROUND: The cell-free RNA (cf-RNA) of spent embryo medium (SEM) has aroused a concern of academic and clinical researchers for its potential use in non-invasive embryo screening. However, comprehensive characterization of cf-RNA from SEM still presents significant technical challenges, primarily due to the limited volume of SEM. Hence, there is urgently need to a small input liquid volume and ultralow amount of cf-RNA library preparation method to unbiased cf-RNA sequencing from SEM. (75) RESULT: Here, we report a high sensitivity agarose amplification-based cf-RNA sequencing method (SEM-Acf) for human preimplantation SEM cf-RNA analysis. It is a cf-RNA sequencing library preparation method by adding agarose amplification. The agarose amplification sensitivity (0.005 pg) and efficiency (105.35 %) were increased than that of without agarose addition (0.45 pg and 96.06 %) by âˆ¼ 90 fold and 9.29 %, respectively. Compared with SMART sequencing (SMART-seq), the correlation of gene expression was stronger in different SEM samples by using SEM-Acf. The cf-RNA number of detected and coverage uniformity of 3' end were significantly increased. The proportion of 5' end adenine, alternative splicing events and short fragments (<400 bp) were increased. It is also found that 4-mer end motifs of cf-RNA fragments was significantly differences between different embryonic stage by day3 spent cleavage medium and day5/6 spent blastocyst medium. (141) SIGNIFICANCE: This study established an efficient SEM amplification and library preparation method. Additionally, we successfully described the characterizations of SEM cf-RNA in preimplantation embryo using SEM-Acf, including expression features and fragment lengths. SEM-Acf facilitates the exploration of cf-RNA as a noninvasive embryo screening biomarker, and opens up potential clinical utilities of small input liquid volume and ultralow amount cf-RNA sequencing. (59).

Association between sarcopenia and kidney stones in United States adult population between 2011 and 2018.

Purpose: To investigate the relationship between kidney stones and sarcopenia in United States adult population between 2011 and 2018. Materials and methods: We conducted a cross-section study based on the National Health and Nutrition Examination Survey (NHANES) including 39,156 individuals. Sarcopenia was assessed by the sarcopenia index. Association between kidney stones and sarcopenia verified by multiple logistic regression analysis and dose-response curves analysis using restricted cubic spline (RCS) regression. Meanwhile, propensity score matching (PSM) was performed to exclude the effect of confounding variables. Results: There were 9,472 participants in the study by our accurate enrollment screening process. The odds of kidney stones decreased significantly with the increase of sarcopenia index. Logistic regression analysis showed that sarcopenia expressed significant differences in the participants which suffered kidney stone before PSM (p < 0.001). In model 4, adjusting all relevant covariates shown that adjusted odds ratio (aOR) of the 95% confidence intervals for kidney stones in all participants, age <39 years and age ≥40 years, were, respectively, 1.286 (1.006-1,643), 1.697 (1.065-2.702), and 0.965 (0.700-1.330) for sarcopenia, and p values were 0.044, 0.026, and 0.827. After performing PSM, the aOR of the 95% in modal 4 for kidney stones in all participants and age <40 year were 2.365 (1.598-3.500) and 6.793 (2.619-17.6180), respectively (p < 0.01), and especially the aOR in participants (age ≥40) was 1.771(1.138-2.757) with p value being 0.011. Conclusion: Sarcopenia was positively related to the potential risk of kidney stones in the United States adult population.

Spatial Transcriptome Profiling of Mouse Hippocampal Single Cell Microzone in Parkinson's Disease.

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and cognitive functions. It is still unknown how gene expression changes in single-cell in different spatial locations of the hippocampus of Parkinson's disease. The purpose of this study was to analyze the gene expression features of single cells in different spatial locations of mouse hippocampus, and to explore the effects of gene expression regulation on learning and memory mechanisms. Here, we obtained 74 single-cell samples from different spatial locations in a mouse hippocampus through microdissection technology, and used single-cell RNA-sequencing and spatial transcriptome sequencing to visualize and quantify the single-cell transcriptome features of tissue sections. The results of differential expression analysis showed that the expression of Sv2b, Neurod6, Grp and Stk32b genes in a hippocampus single cell at different locations was significantly different, and the marker genes of CA1, CA3 and DG subregions were identified. The results of gene function enrichment analysis showed that the up-regulated differentially expressed genes Tubb2a, Eno1, Atp2b1, Plk2, Map4, Pex5l, Fibcd1 and Pdzd2 were mainly involved in neuron to neuron synapse, vesicle-mediated transport in synapse, calcium signaling pathway and neurodegenerative disease pathways, thus affecting learning and memory function. It revealed the transcriptome profile and heterogeneity of spatially located cells in the hippocampus of PD for the first time, and demonstrated that the impaired learning and memory ability of PD was affected by the synergistic effect of CA1 and CA3 subregions neuron genes. These results are crucial for understanding the pathological mechanism of the Parkinson's disease and making precise treatment plans.

Cell-Free DNA Fragmentomics: The Novel Promising Biomarker.

Cell-free DNA molecules are released into the plasma via apoptotic or necrotic events and active release mechanisms, which carry the genetic and epigenetic information of its origin tissues. However, cfDNA is the mixture of various cell fragments, and the efficient enrichment of cfDNA fragments with diagnostic value remains a great challenge for application in the clinical setting. Evidence from recent years shows that cfDNA fragmentomics' characteristics differ in normal and diseased individuals without the need to distinguish the source of the cfDNA fragments, which makes it a promising novel biomarker. Moreover, cfDNA fragmentomics can identify tissue origins by inferring epigenetic information. Thus, further insights into the fragmentomics of plasma cfDNA shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In this review, we focus on the cfDNA fragment characteristics and its potential application, such as fragment length, end motifs, jagged ends, preferred end coordinates, as well as nucleosome footprints, open chromatin region, and gene expression inferred by the cfDNA fragmentation pattern across the genome. Furthermore, we summarize the methods for deducing the tissue of origin by cfDNA fragmentomics.

A comprehensive characterization of cell-free RNA in spent blastocyst medium and quality prediction for blastocyst.

The rate of pregnancy can be affected by many factors in assisted reproductive technology (ART), and one of which is the quality of embryos. Therefore, selecting the embryos with high potential is crucial for the outcome. Fifteen spent blastocyst medium (SBM) samples were collected from 14 patients who received in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), seven from high-grade embryos and eight from low-grade embryos. Cell-free RNA (cf-RNA) profile of SBM samples were analyzed by RNA sequencing in the present study. It was found that a large amount of cf-RNA were released into SBM, including protein-coding genes (68.9%) and long noncoding RNAs (lncRNAs) (17.26%). Furthermore, a high correlation was observed between blastocyst genes and SBM genes. And the cf-mRNAs of SBM were highly fragmented, and coding sequence (CDS) and untranslated (UTR) regions were released equally. Two hundred and thirty-two differentially expressed genes were identified in high-grade SBM (hSBM) and low-grade SBM (lSBM), which could be potential biomarker in distinguishing the embryos with different quality as an alternative or supplementary approach for subjective morphology criteria. Hence, cf-RNAs sequencing revealed the characterization of circulating transcriptomes of embryos with different quality. Based on the results, the genes related to blastocyst quality were screened, including the genes closely related to translation, immune-signaling pathway, and amino acid metabolism. Overall, the present study showed the types of SBM cf-RNAs, and the integrated analysis of cf-RNAs profiling with morphology grading displayed its potential in predicting blastocyst quality. The present study provided valuable scientific basis for noninvasive embryo selection in ART by RNA-profiling analysis.

LncRNA SNHG3 enhances BMI1 mRNA stability by binding and regulating c-MYC: Implications for the carcinogenic role of SNHG3 in bladder cancer.

The transformation of nonmuscle-invasive bladder cancer (BLCa) to muscle-invasive type and distant metastasis are the two major threats to patients after surgery. Thus, it is important to identify the key genes of BLCa cell invasion and metastasis. Long noncoding RNA (lncRNA) is a potential clinical tool for cancer diagnosis and treatment. Herein, we verified that lncRNA SNHG3 is upregulated in human BLCa specimens and is proportional to poor clinical prognosis via a combination of bioinformatic analyses and wet bench experiments. Then, we constructed SNHG3 knockdown and overexpression cell models via lentiviral packaging and CRISPR-Cas9 technique. Fluorescence in situ hybridization assay showed that SNHG3 is distributed in both the nucleus and cytoplasm of BLCa cell lines. In vitro assays including CCK-8, EdU, colony formation, wound healing, transwell, and tube formation demonstrated that SNHG3 knockdown and overexpression potently inhibited and enhanced BLCa cell proliferation, migration, invasion, and angiogenesis. In addition, IVIS imaging revealed that SNHG3 knockdown could significantly inhibit M-NSG mice xenograft tumor growth. Next, RNA sequencing, bioinformatics analyses and western blots indicated that SNHG3 could promote c-MYC expression. RNA immunoprecipitation, actinomycin D assay and western blot assays suggested that SNHG3 could also bind c-MYC protein which subsequently facilitate the stabilization of BMI1 mRNA, thus enhancing BMI1 protein level. However, SNHG3 knockdown had a slightly weaker inhibitory effect on BMI1 expression than c-MYC knockdown. Further, in vitro assays demonstrated that BMI1 knockdown could suppress the SNHG3 activation-induced tumor promoting effect in BLCa cells. Overall, this study has provided new insights into the potential implication of lncRNA SNHG3 in the pathogenesis of BLCa. Importantly, SNHG3/c-MYC/BMI1 axis may be a novel target for regulating tumor growth and metastasis in BLCa patients.

Extracellular cell-free RNA profile in human large follicles and small follicles.

Background: Previous studies have shown that a large number of valuable and functional cell-free RNAs (cfRNAs) were found in follicular fluid. However, the species and characteristics of follicular fluid cfRNAs have not been reported. Furthermore, their implications are still barely understood in the evaluation of follicular fluid from follicles of different sizes, which warrants further studies. Objective: This study investigated the landscape and characteristics of follicular fluid cfRNAs, the source of organization, and the potential for distinguishing between follicles of different sizes. Methods: Twenty-four follicular fluid samples were collected from 20 patients who received in vitro fertilization (n = 9) or ICSI (n = 11), including 16 large follicular fluid and 8 small follicular fluid samples. Also, the cfRNA profile of follicular fluid samples was analyzed by RNA sequencing. Results: This result indicated that the concentration of follicular fluid cfRNAs ranged from 0.78 to 8.76 ng/ml, and fragment length was 20-200 nucleotides. The concentration and fragment length of large follicular fluid and small follicular fluid samples were not significantly different (p > 0.05). The technical replica correlation of follicular fluid samples ranged from 0.3 to 0.9, and the correlation of small follicular fluid samples was remarkably (p < 0.001) lower than that of large follicular fluid samples. Moreover, this study found that cfRNAs of the follicular fluid could be divided into 37 Ensembl RNA biotypes, and a large number of mRNAs, circRNAs, and lncRNAs were observed in the follicular fluid. The number of cfRNAs in large follicular fluid was remarkably (p < 0.05) higher than that of small follicular fluid. Furthermore, the follicular fluid contained a large amount of intact mRNA and splice junctions and a large number of tissue-derived RNAs, which are at a balanced state of supply and elimination in the follicular fluid. KEGG pathway analysis showed that differentially expressed cfRNAs were enriched in several pathways, including thyroid hormone synthesis, the cGMP-PKG signaling pathway, and inflammatory mediator regulation of TRP channels. In addition, we further showed that four cfRNAs (TK2, AHDC1, PHF21A, and TTYH1) serve as a potential indicator to distinguish the follicles of different sizes. The ROC curve shows great potential to predict follicular fluid from follicles of different sizes [area under the curve (AUC) > 0.88]. Conclusion: Overall, our study revealed that a large number of cfRNAs could be detected in follicular fluid and could serve as a potential non-invasive biomarker in distinguishing between follicles of different sizes. These results may inform the study of the utility and implementation of cfRNAs in clinical practice.

Prediction of Time-Series Transcriptomic Gene Expression Based on Long Short-Term Memory with Empirical Mode Decomposition.

RNA degradation can significantly affect the results of gene expression profiling, with subsequent analysis failing to faithfully represent the initial gene expression level. It is urgent to have an artificial intelligence approach to better utilize the limited data to obtain meaningful and reliable analysis results in the case of data with missing destination time. In this study, we propose a method based on the signal decomposition technique and deep learning, named Multi-LSTM. It is divided into two main modules: One decomposes the collected gene expression data by an empirical mode decomposition (EMD) algorithm to obtain a series of sub-modules with different frequencies to improve data stability and reduce modeling complexity. The other is based on long short-term memory (LSTM) as the core predictor, aiming to deeply explore the temporal nonlinear relationships embedded in the sub-modules. Finally, the prediction results of sub-modules are reconstructed to obtain the final prediction results of time-series transcriptomic gene expression. The results show that EMD can efficiently reduce the nonlinearity of the original data, which provides reliable theoretical support to reduce the complexity and improve the robustness of LSTM models. Overall, the decomposition-combination prediction framework can effectively predict gene expression levels at unknown time points.

Sensitive and Low-Bias Transcriptome Sequencing Using Agarose PCR.

Transcriptome sequencing has emerged as an important research tool for exploring the mysteries of life at the single-cell level. However, its wide application is limited by the bias associated with the amplification reactions which is essential for library building of trace RNA. In this study, low-melting-point agarose was added to the amplification reactions to take advantage of its molecular crowding effect and polymer cross-linked structure to improve the sensitivity of the reactions and reduce bias. To further evaluate the performance of the method, it was applied to transcriptome sequencing of microregion samples from brain tissue sections of mice with Parkinson's disease at the single cell level. The results showed that agarose PCR had better performance than in-tube PCR. Further application of agarose PCR to transcriptome library sequencing could obtain data closer to that of unamplified. With the addition of low melting point agarose, the sensitivity of the amplification reaction was significantly increased, while homogeneity was increased by approximately 2-fold. Not only that, but this work also provides 11% sensitivity improvement for spatial transcriptomic study on Parkinson's disease-associated gene detection. The agarose PCR provides a new tool for efficient and homogeneous amplification of trace samples and can be widely used for spatial transcriptome library sequencing and studies.

Integrity of cell-free DNA in maternal plasma extracellular vesicles as a potential biomarker for non-invasive prenatal testing.

OBJECTIVE: Large proportions of cell-free DNA (cfDNA) in plasma are localized in extracellular vesicles (EVs), which are secreted from placental cells. This study was conducted to reveal the integrity pattern of cfDNA in maternal plasma EVs (evcfDI) across gestation, and explore if evcfDI could be a potential biomarker in screening for aneuploid fetus in non-invasive prenatal testing (NIPT). METHODS: A total of 180 maternal plasma samples were collected during NIPT. Both evcfDNA and fetal evcfDNA (evcffDNA) were measured by quantitative PCR of LINE1 and SRY gene amplicons with different sizes. The evcfDI was calculated as the ratio of long to short fragments. RESULTS: evcfDI is not affected by gestational age; whereas evcffDI has a mild decreasing trend with increasing gestational age (P = 0.048). evcfDI is significantly and negatively correlated with maternal body mass index (BMI; calculated as weight in kilograms divided by the square of height in meters: ≤18.5, 18.5-25, and ≥25) (P < 0.01) and age (<35 and ≥35 years) (P < 0.01). Mean evcfDI decreases from 2.113 in euploid controls to 0.681 in those with an aneuploid fetus in NIPT (P = 0.003). CONCLUSION: Maternal clinical characteristics such as BMI and age could be innovative biomarkers to calibrate evcfDI, which was shown to be a potential indicator of an aneuploid fetus. Analysis of evcfDI based on quantitative PCR could serve as a novel, rapid, and low-cost NIPT strategy, which might facilitate testing at earlier gestations.

Comparative analysis of circular RNA enrichment methods.

The circRNAs sequencing results vary due to the different enrichment methods and their performance is needed to systematic comparison. This study investigated the effects of different circRNA enrichment methods on sequencing results, including abundance and species of circRNAs, as well as the sensitivity and precision. This experiment was carried out by following four common circRNA enrichment methods: including ribosomal RNA depletion (rRNA-), polyadenylation and poly (A+) RNA depletion followed by RNase R treatment (polyA+RNase R), rRNA-+polyA+RNase R and polyA+RNase R+ rRNA-. The results showed that polyA+RNase R+ rRNA - enrichment method obtained more circRNA number, higher sensitivity and abundance among them; polyA+RNase R method obtained higher precision. The linear RNAs can be thoroughly removed in all enrichment methods except rRNA depletion method. Overall, our results helps researchers to quickly selection a circRNA enrichment of suitable for own study among many enrichment methods, and it provides a benchmark framework for future improvements circRNA enrichment methods.[Figure: see text].

Combination of C-Reactive Protein and Neutrophil-to-Lymphocyte Ratio as a Novel Prognostic Index in Patients With Bladder Cancer After Radical Cystectomy.

BACKGROUND: Inflammation is widely considered an important hallmark of cancer and associated with poor postoperative survival. The objective of this study is to assess the significance of preoperative C-NLR, a new inflammation-based index that includes preoperative C-reactive protein (CRP) and neutrophil-to-lymphocyte ratio (NLR), on therapeutic outcomes for bladder cancer (BC) patients after radical cystectomy (RC). MATERIALS AND METHODS: BC patients who underwent RC between 2010 and 2019 were retrospectively analyzed from our medical center. The predictive effect of CRP, NLR, and C-NLR on the survival of BC patients were analyzed by the receiver operating characteristic (ROC) curves. The relationship between C-NLR and postoperative survival was investigated by Cox regression. The corresponding nomograms were built based on the Cox regression results of overall survival (OS) and disease-free survival (DFS), which were further validated by ROC curves, decision curve analysis (DCA) curves, and calibration curves. RESULTS: Of the 199 eligible patients, 83 (41.70%) were classified as high C-NLR group and the remaining 116 (58.30%) were classified as low C-NLR group. ROC analysis showed that C-NLR had the largest area under curve (AUC) compared to CRP and NLR. Multivariate analysis revealed that T-stage and C-NLR [high C-NLR vs. low C-NLR, hazard ratio (HR) = 2.478, 95% confidence interval (CI), 1.538-3.993, p < 0.001] were independent predictors of OS, whereas T-stage, M-stage, and C-NLR (high C-NLR vs. low C-NLR, HR = 2.817, 95% CI, 1.667-4.762, p < 0.001) were independent predictors of DFS. ROC and DCA analysis demonstrated better accuracy and discrimination of 3- and 5-year OS and DFS with C-NLR-based nomogram compared to TNM stage. The calibration curve reconfirmed the accurate predicting performance of nomograms. CONCLUSION: C-NLR is a reliable predictor of long-term prognosis of BC patients after RC and will contribute to the optimization of individual therapy for BC patients.

The integrity of cfDNA in follicular fluid and spent medium from embryo culture is associated with embryo grade in patients undergoing in vitro fertilization.

PURPOSE: This study was conducted to verify if the cfDNA integrity (cfDI) in follicular fluid and subsequent spent embryo medium (SEM) could serve as potential non-invasive biomarker for high-grade embryo selection during IVF/ICSI. METHODS: Thirty-two follicular fluids, 32 subsequent corresponding cleavage embryo SEM, and 23 subsequent blastocyst SEM were collected from 11 patients undergoing IVF/ICSI. CfDI was measured by ALU gene amplicons with different sizes by qPCR, as the ratio of long to short fragments. RESULTS: CfDI in follicular fluid corresponding to subsequent high-grade cleavage embryos and blastocysts was significantly lower than that related to low-grade embryos (p = 0.018). Conversely, cfDI in SEM was significantly and positively correlated with high-grade embryos at both stages (p = 0.009). ROC curves of the analysis of cfDI in follicular fluid showed great potential in predicting subsequent embryogenesis and embryo grade (AUC > 0.927). Regardless of the cleavage embryo grade by morphology, cfDI in day 3 SEM could predict if the cleavage embryo could develop to a high-grade blastocyst (AUC = 0.820). A concordant shift pattern of cfDI from follicular fluid to subsequent day 3 SEM and day 5 SEM was found in 81.82% participants featured by various clinical characteristics. CONCLUSION: CfDI in follicular fluid and SEM was significantly correlated with embryogenesis and embryo grade and could serve as a potential non-invasive biomarker in high-grade embryo selection. Direct qPCR was proved as a labor-saving and sensitive method for the analysis of cfDI in low volume of SEM.

Prognostic Value of Lymphocyte-C-Reactive Protein Ratio in Patients Undergoing Radical Cystectomy for Bladder Cancer: A Population-Based Study.

PURPOSE: This study aimed to assess the prognostic value of the lymphocyte-C-reactive protein ratio (LCR) in patients with bladder cancer (BCa) who underwent radical cystectomy (RC). MATERIALS AND METHODS: BCa patients between 2009 and 2018 were retrieved from our medical center. The predictive value of LCR on survival of BCa patients was evaluated through the Kaplan-Meier survival and receiver operating characteristic (ROC) curves. The multivariate Cox regression results were used for conducting the nomogram, which were further verified by ROC, decision curve analysis (DCA), and calibration curves. Propensity score matching (PSM) was performed to validate our findings. RESULTS: A total of 201 BCa patients who received RC were included in this study, with 62 (30.8%) patients in the low LCR group and 139 (69.2%) in the high LCR group. Multivariate analysis results revealed that the high LCR group was significantly related to better prognosis and functioned as a prognostic biomarker for overall survival (OS) [hazard ratio (HR) = 0.41, 95% CI, 0.26-0.66; p < 0.001] and disease-free survival (DFS) [HR = 0.40, 95% CI, 0.26-0.66; p < 0.001]. The nomogram processed better predictive capability and accuracy than TNM stage from ROC results (AUC = 0.754 vs. AUC = 0.715), with the confirmation of calibration curves and DCA. The result of PSM confirmed that LCR was significantly correlated with OS and DFS. CONCLUSION: Our finding demonstrates that LCR is a novel, convenient, and effective predictor that may provide vital assistance for clinical decision and individualized therapy in BCa patients after RC.

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing. RESULTS: Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified. CONCLUSIONS: The ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.

Low bias multiple displacement amplification with confinement effect based on agarose gel.

Multiple displacement amplification (MDA) is a popular single-cell whole-genome amplification (WGA) technique that can greatly improve the amplification efficiency of single-cell genomes. However, there is an inherent problem that cannot be completely solved, that is, the amplification bias. We here propose an improved MDA method based on low melting agarose gel, named gelMDA. Firstly, the agarose gel and solution were characterized with SEM and fluorescent reagent. Then, we used gelMDA for cDNA amplification in library preparation of RNA-seq, and conventional MDA was used as a comparison. The sensitivity, efficiency of gelMDA, and amplification bias were evaluated with fluorescence curve, product yield, and the sequencing results. Finally, gelMDA was used for single-cell transcriptome sequencing. The results showed that the sensitivity and product yield of gelMDA were significantly higher than those of conventional MDA. A lower coefficient of variation (CV) and a higher reproducibility were obtained from gelMDA sequencing results. A region of 30 µm in diameter was amplified from the tissue sections and successfully sequenced. In conclusion, gelMDA obtained higher amplification efficiency and lower amplification bias in the present study. It suggested the great potential in single-cell RNA amplification and sequencing.

Bias in RNA-seq Library Preparation: Current Challenges and Solutions.

Although RNA sequencing (RNA-seq) has become the most advanced technology for transcriptome analysis, it also confronts various challenges. As we all know, the workflow of RNA-seq is extremely complicated and it is easy to produce bias. This may damage the quality of RNA-seq dataset and lead to an incorrect interpretation for sequencing result. Thus, our detailed understanding of the source and nature of these biases is essential for the interpretation of RNA-seq data, finding methods to improve the quality of RNA-seq experimental, or development bioinformatics tools to compensate for these biases. Here, we discuss the sources of experimental bias in RNA-seq. And for each type of bias, we discussed the method for improvement, in order to provide some useful suggestions for researcher in RNA-seq experimental.

Utilization of pelleted and extruded feed by blunt snout bream Megalobrama amblycephala: Insights from growth performance, health status and feed cost.

The present study was conducted to evaluate the utilization of both pelleted feed (PF) and extruded feed (EF) by blunt snout bream Megalobrama amblycephala based on growth performance, stress responses, innate immunity and disease resistance. Both the PF and EF were prepared with the same formula. Fish were divided randomly into 2 groups, including one fed the PF continuously and one offered the EF continuously. The whole feeding trial lasted 8 weeks, after which fish were subjected to Aeromonas hydrophila infection. The results showed that the feed intake, feed conversion ratio, hepatic total superoxide dismutase activity and glutathione content, plasma complement 3 and complement 4 levels as well as myeloperoxidase activity of the EF group were all significantly lower than those of the PF group, while the opposite was true for the condition factor, the viscera index, the abdominal fat percentage, nitrogen and energy retention, hepatic malondialdehyde content, plasma levels of cortisol, glucose, lactate, total protein and globulin as well as the activities of plasma alanine aminotransferase and aspartate aminotransferase. In addition, the EF group also obtained relatively low activities of hepatic glutathione peroxidase and plasma acid phosphatase as well as high cumulative mortality rates at 24-96 h after Aeromonas hydrophila challenge. Furthermore, the feed cost of culturing this species with EF is lower than that with PF. These findings indicated that compared with PF, EF could increase the feed utilization and economic benefits of blunt snout bream, but reduce its anti-stress ability, non-specific immunity, A. hydrophila resistance and feed cost.