Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Schematic representation of mechanistic strategies of barcoding. (A–C) Barcodes can be introduced to a template using adaptors through direct ligation (A) , using RT- or PCR primers at the reverse transcription or PCR amplification step (B) , and using hybridizing molecular inversion probes (C) . (D) Schematic representation of the difference between “barcodes” and “sample indexes”. Barcodes aim to correct sequencing errors. For example, a misreading nucleotide, guanosine (G) can be corrected in final consensus sequences for a pool of Sample 1 (top panel). Sample indexes are used to multiplex different sequencing amplicons generated from different pools of samples (Sample 1, 2, and 3) (bottom panel). Panel (A) is modified based on in and panel (C) is modified based on in .

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Ligation, Reverse Transcription, Amplification, Sequencing, Multiplex Assay, Generated, Modification

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Comparison, Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Extraction, Ligation, DNA Sequencing, Multiplexing, Generated, Reverse Transcription, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection

Journal: Journal of Biological Chemistry

Article Title: Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis

doi: 10.1074/jbc.m309338200

Figure Lengend Snippet: FIG. 6. Functional characterization of AtOPT3. A, hybridization of an 600-bp AtOPT3 cDNA probe, corresponding to the 3-terminal exon, to RNA extracted from Arabidopsis roots of plants grown in control conditions (Ctl), copper (-Cu), manganese (-Mn), zinc (-Zn), and iron (-Fe) deficiency. The ethidium bromide-stained RNA gel is shown for quantification. B, growth of ctr1 expressing AtOPT3 on YPG-Ura plates supplemented with 10 M CuSO4 compared with the mutant transformed with the vector alone. C, growth of smf1 expressing AtOPT3 on manganese-limited medium, with and without 1 mM EGTA compared with the growth of the mutant transformed with the vector alone.

Article Snippet: Genome-wide Analysis Provides Insight into Metal Transport—We have used Affymetrix Arabidopsis DNA chips containing 8,300 genes (which cover about one-third of the ge- FIG. 7.

Techniques: Functional Assay, Hybridization, Control, Staining, Expressing, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: Nutrients

Article Title: Long-Term Consumption of a Sugar-Sweetened Soft Drink in Combination with a Western-Type Diet Is Associated with Morphological and Molecular Changes of Taste Markers Independent of Body Weight Development in Mice

doi: 10.3390/nu14030594

Figure Lengend Snippet: Transcriptome analysis of 59 selected genes associated with oral chemosensation, displayed as a heatmap showing mean fold changes in gene expression of the SSB-fed diet groups in relation to the respective water-fed group (=1) in the form of a color code. The gene expression was analyzed using one customized cDNA microarray per group from pooled RNA samples of the CV from mice that received either a standard diet (chow, n = 10–11) or Western-type diet (WD, n = 7–8) with water (Water) or a sugar-sweetened beverage (SSB) as a drink for 24 weeks.

Article Snippet: The isolated RNA samples per mouse were reverse transcribed to cDNA using the LunaScript RT Supermix Kit (New England Biolabs GmbH, Frankfurt am Main, Germany).

Techniques: Expressing, Microarray, Western Blot

Journal: PLoS ONE

Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

doi: 10.1371/journal.pone.0194036

Figure Lengend Snippet: If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).

Article Snippet: DNA from patients was digested using a cocktail of three MSREs: HpaII (5′-CˆCGG-3′), HhaI (5′-GCGˆC-3′), and AciI (5′-CˆCGC-3′) (New England BioLabs, Ipswich, USA), which in combination interrogate the methylation status of ~32.4% of CpG sites in the human genome [ ].

Techniques: DNA Methylation Assay, Microarray, Methylation

Journal: PLoS ONE

Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

doi: 10.1371/journal.pone.0194036

Figure Lengend Snippet: SNP, single-nucleotide polymorphism; MSRE, methylation-sensitive restriction enzyme; RAS, relative allele score; IBD, inflammatory bowel disease; (G), genomic DNA; (D), DNA digested MSREs; (U), whole-genome-amplified fully unmethylated DNA digested MSREs.

Article Snippet: DNA from patients was digested using a cocktail of three MSREs: HpaII (5′-CˆCGG-3′), HhaI (5′-GCGˆC-3′), and AciI (5′-CˆCGC-3′) (New England BioLabs, Ipswich, USA), which in combination interrogate the methylation status of ~32.4% of CpG sites in the human genome [ ].

Techniques: Methylation, Amplification

Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA . ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Reverse Transcription, Purification, Amplification, In Vitro

Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Experimental procedure utilized for the analysis of 10-year-old matched frozen and formalin-fixed paraffin embedded breast cancer samples. ( a ) Five micrograms of RNA extracted from the 10-year-old frozen portion of the sample, is reverse-transcribed and the cDNA is double stranded (dsDNA), in four individual reactions. The dsDNA of three reactions undergoes IVT-amplification (MessageAmpII, Ambion), which gives rise to complementary RNA (cRNA) for cDNA microarray analyses. The dsDNA of one reaction is used for PCR experiments. ( b ) Five micrograms of RNA extracted from the 10-year-old FFPE portion of the sample underwent the exact same process. ( c ) Single-stranded DNA (ssDNA) obtained by RT of 5 μg of FFPE-RNA is purified and hybridized to the sense-RNA template library. The restored ssDNA is double stranded and purified. Three of the CT-RT reactions undergo IVT-amplification, while the dsDNA of one reaction is used for PCR experiments.

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Formalin-fixed Paraffin-Embedded, Reverse Transcription, Amplification, Microarray, Purification

Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Signal intensity and heat-map analysis of the correlation between the log 2 ratios measured by cDNA microarrays. ( a ) Signal intensity of one sample grid in the red channel (Cy5) across all microarrays. Top three panels display the grids obtained from three repeats using cRNA from 10-year-old frozen RNA (Frozen-Amp 1–3). Three mid-panels show the signal of three repeats using cRNA obtained by restoration and IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Restored 1–3). Three bottom panels display the signal of three repeats using cRNA obtained by direct IVT-amplification of RNA from 10-year-old FFPE tissue. ( b ) Heat map displaying the log 2 of expression ratios ranging between 0.5 and 2 for 1044 genes detected in frozen tissue on a 28 032 features cDNA microarray and represented in the UHR library. From left to right are displayed the ratios obtained by IVT-amplification of RNA from 10-year-old frozen tissue (Frozen-Amp 1–3), restoration and IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Restored 1–3) and direct IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Amp 1–3). Each column represents an individual hybridization and each line a different feature. Red and blue represent up-regulated and down-regulated genes, respectively.

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Amplification, Expressing, Microarray, Hybridization