Showing papers on "Complementary DNA published in 2022"

Single-cell isoform analysis in human immune cells

Abstract: High-throughput single-cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5' or 3' end. Here, we used R2C2 to sequence and demultiplex 12 million full-length cDNA molecules generated by the 10X Genomics platform from ~3000 peripheral blood mononuclear cells. We use these reads, independent from Illumina data, to identify B cell, T cell, and monocyte clusters and generate isoform-level transcriptomes for cells and cell types. Finally, we extract paired adaptive immune receptor sequences unique to each T and B cell.

Single-cell isoform analysis in human immune cells

Abstract: High-throughput single-cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5' or 3' end. Here, we used R2C2 to sequence and demultiplex 12 million full-length cDNA molecules generated by the 10X Genomics platform from ~3000 peripheral blood mononuclear cells. We use these reads, independent from Illumina data, to identify B cell, T cell, and monocyte clusters and generate isoform-level transcriptomes for cells and cell types. Finally, we extract paired adaptive immune receptor sequences unique to each T and B cell.

Lateral flow–based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use

Abstract: The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.

Direct RNA targeted in situ sequencing for transcriptomic profiling in tissue

Abstract: Highly multiplexed spatial mapping of transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing chemistry. We benchmark a High Sensitivity Library Preparation Kit from CARTANA that circumvents the reverse transcription needed for cDNA-based in situ sequencing (ISS) via direct RNA detection. We found a fivefold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell clustering. With this increased efficiency, we also found to maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap to other fluorescent in situ hybridization-based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS.

Plug-in label-free optical fiber DNA hybridization sensor based on C-type fiber Vernier effect

Abstract: A reflective Fabry-Perot interference (FPI) label-free biosensor based on Vernier effect is designed and implemented for in situ real-time DNA hybridization detection. The sensor is made by splicing single-mode fiber (SMF), a segment of C-type fiber and SMF. The Vernier effect occurs between the solid cavity FPI and the air cavity FPI. In our experiment, a high sensitivity of 10791.12 nm/RIU is obtained in the refractive index (RI) range of 1.3311–1.3450. On this basis, we immobilized probe DNA (pDNA) on the surface of SMF to detect the complementary DNA (cDNA), demonstrating use for label-free sensing of DNA hybridization. The experimental results show that the sensor can detect 1 µM cDNA with high specificity. The paper demonstrates the first research of optical fiber DNA biosensor based on the open microcavity FPI Vernier effect. In addition, the sensor has the advantages of high sensitivity, strong stability, simple structure and simple fabrication. It can also be applied to other biomolecule detection based on probe-target biological binding events, which has great application potential in medical diagnostics, food science, genetic engineering and other biological fields.

Isolation and Functional Characterization of a LEAFY Gene in Mango (Mangifera indica L.)

Abstract: LEAFY (LFY) plays an important role in the flowering process of plants, controlling flowering time and mediating floral meristem differentiation. Owing to its considerable importance, the mango LFY gene (MiLFY; GenBank accession no. HQ585988) was isolated, and its expression pattern and function were characterized in the present study. The cDNA sequence of MiLFY was 1152 bp, and it encoded a 383 amino acid protein. MiLFY was expressed in all tested tissues and was highly expressed in flowers and buds. Temporal expression analysis showed that MiLFY expression was correlated with floral development stage, and two relative expression peaks were detected in the early stages of floral transition and floral organ differentiation. Moreover, 35S::GFP-MiLFY fusion protein was shown to be localized to the nucleus of cells. Overexpression of MiLFY in Arabidopsis promoted early flowering and the conversion of lateral meristems into terminal flowers. In addition, transgenic plants exhibited obvious morphological changes, such as differences in cauline leaf shape, and the number of lateral branches. When driven by the MiLFY promoter, GFP was highly expressed in leaves, floral organs, stems, and roots, during the flowering period. Exogenous gibberellin (GA3) treatment downregulated MiLFY promoter expression, but paclobutrazol (PPP333) upregulated it. Bimolecular fluorescence complementation (BiFC) assays showed that the MiLFY protein can interact with zinc-finger protein 4 (ZFP4) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (MiSOC1D). Taken together, these results indicate that MiLFY plays a pivotal role in controlling mango flowering, and that it is regulated by gibberellin and paclobutrazol.

Dual-signal electrochemical aptasensor involving hybridization chain reaction amplification for aflatoxin B1 detection

Abstract: Since aflatoxin B1 (AFB1) is a highly toxic and carcinogenic secondary metabolite, it is of great importance to develop an effective method for detection of it. Based on hybridization chain reaction (HCR), we proposed a ratiometric dual-signal electrochemical aptasensor (DECA) to realize rapid and sensitive detection of AFB1. In the aptasensor, ferrocene (Fc) and methylene blue (MB)-tagged single-stranded DNA sequences acted as dual-signal sources. Based on this, more stable hairpin DNA (HS-H1) and MB-labeled auxiliary hairpin DNA (H1 and H2) were introduced to extend the sensing platform. In presence of AFB1, it will compete with MB-cDNA and specifically combine with Fc-labeled aptamer. As a result, MB-cDNA will release and trigger the HCR for MB signal amplification with an enhancement in sensitivity. Besides, the ratio of current variations behaved a ratiometric signal output mode. In the proof-of-concept study, the single-signal mode behaved a linear range of 1 pM~10 nM with limit of detection (LOD) of 0.56 pM (IMB) and 0.15 pM (IFc), respectively, while the ratiometric detection mode showed the lowest LOD of 0.12 pM (S/N = 3). The good specificity, reproducibility, and stability of DECA further revealed its promising application in food safety monitoring.

A versatile 5′ RACE-Seq methodology for the accurate identification of the 5′ termini of mRNAs

Abstract: Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. However, 5' ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The implementation of the template-switching mechanism at the reverse transcription stage along with 5' rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5' ends of cDNAs. In the current study, we developed a 5' RACE-seq method by coupling a custom template-switching and 5' RACE assay with targeted nanopore sequencing, to accurately unveil 5' termini of mRNA targets.The optimization of the described 5' RACE-seq method was accomplished using the human BCL2L12 as control gene. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5' RACE. Collectively, our results support the existence of two distinct 5' termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. As proof of concept, we implemented the described 5' RACE-seq methodology to investigate the 5' UTRs of several kallikrein-related peptidases (KLKs) gene family members. Our results confirmed the existence of multiple annotated 5' UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones.In this work we present an in-house developed 5' RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. This approach enables the broad and in-depth study of 5' UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits.

A versatile 5′ RACE-Seq methodology for the accurate identification of the 5′ termini of mRNAs

Abstract: Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. However, 5' ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The implementation of the template-switching mechanism at the reverse transcription stage along with 5' rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5' ends of cDNAs. In the current study, we developed a 5' RACE-seq method by coupling a custom template-switching and 5' RACE assay with targeted nanopore sequencing, to accurately unveil 5' termini of mRNA targets.The optimization of the described 5' RACE-seq method was accomplished using the human BCL2L12 as control gene. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5' RACE. Collectively, our results support the existence of two distinct 5' termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. As proof of concept, we implemented the described 5' RACE-seq methodology to investigate the 5' UTRs of several kallikrein-related peptidases (KLKs) gene family members. Our results confirmed the existence of multiple annotated 5' UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones.In this work we present an in-house developed 5' RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. This approach enables the broad and in-depth study of 5' UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits.

Ratiometric electrochemical aptasensor for point-of-care testing Vibrio parahaemolyticus together with antimicrobial peptide-labeled nano metal-organic framework signal tag

Abstract: It is critically important to develop assays for point-of-care testing (POCT) vibrio in water. In this study, an aptamer-modified electrode was used in conjunction with an antimicrobial peptide-labeled nano metal-organic framework (NMOF) signal tag to screen Vibrio parahaemolyticus (VP) as a model. Firstly, single stranded DNA labeled methylene blue (cDNA-MB) was hybridized with VP aptamer (Apt) on the electrode to form an electroactive DNA probe. Simultaneously, NMOF modified antimicrobial peptide and ferrocene ([email protected]) was prepared as signal tag. The aptasensor was incubated in VP samples that would compete with aptamer and release the cDNA-MB into the supernatant. Afterwards, the [email protected] signal tag was conjugated with the captured VP to form a sandwich complex, resulting in the generation of an electrical signal from Fc. The cDNA-MB signal (IMB) decreased and the Fc signal (IFc) accordingly increased as VP concentration increased. The IFc/IMB ratio was proportional to the logarithm value of VP concentration, with a lower limit of detection as 4 CFU·mL-1. The whole analysis was completed within 30 min with high sensitivity and specificity, which was due to the dual recognition from antimicrobial peptide and aptamer then the amplification of NMOF. The aptasensor could on-site monitor vibrio contamination in aquaculture water.