Journal: Journal of Neuro-Oncology

Article Title: MGMT promoter methylation status for glioblastoma: defining the clinically relevant cut-off value for pyrosequencing

doi: 10.1007/s11060-026-05633-0

Figure Lengend Snippet: Flowchart of patient selection. MGMTpm, MGMT promoter methylation; RT, radiotherapy; TMZ, Temozolomide. * Meningioma, ependymoma, diffuse midline glioma, pilocytic astrocytoma, ganglioglioma, fibrillary astrocytoma, oligodendroglioma IDHmut, gemistocytic astrocytoma IDHmut and germinoma ** Including patients not receiving postoperative treatment. *** preopPS, or extent of resection **** thereby absent from the SQR

Article Snippet: A third option is to establish a cut-off based on outcome, such as in the study by Quillien where the Pyromark MGMT kit investigating CpGs 74–78 in exon 1 of the MGMT gene by pyrosequencing was used and a cut-off of ≥ 9% for mMGMT was proposed [ ].

Techniques: Selection, Methylation

Journal: Journal of Neuro-Oncology

Article Title: MGMT promoter methylation status for glioblastoma: defining the clinically relevant cut-off value for pyrosequencing

doi: 10.1007/s11060-026-05633-0

Figure Lengend Snippet: MGMT promoter methylation status and cut-offs. ( a ) Distribution of mean MGMTpm status of all 451 patients. The solid green line representing the supervised cut-off ( b ) and the dashed black line representing the unsupervised cut-off ( c ). Distributions for each individual CpG are presented in Supplementary Fig. . ( b ) Cut-off supervised by OS and adjusted for prognostic factors: age, extent of resection and preoperative ECOG performance status (solid green line), determined through 5-fold cross-validation with the highest concordance index corresponding to the best performing AFT (accelerated failure time) model. ( c ) Bimodal normal mixture model, with unsupervised cut-off (dashed black line) determined as the intersection point of the two distributions. Blue bars represent patients with methylated MGMT and red bars represent patients with unmethylated MGMT, according to the supervised cut-off (solid green line, b )

Article Snippet: A third option is to establish a cut-off based on outcome, such as in the study by Quillien where the Pyromark MGMT kit investigating CpGs 74–78 in exon 1 of the MGMT gene by pyrosequencing was used and a cut-off of ≥ 9% for mMGMT was proposed [ ].

Techniques: Methylation, Biomarker Discovery

Journal: bioRxiv

Article Title: Unbiased Long-Read Whole-Genome Sequencing Enables High-Resolution Mapping of Transgene Concatenation and Off-target Genomic Disruption in a Mouse Model

doi: 10.64898/2026.05.15.725597

Figure Lengend Snippet: (A) Schematic showing three regions of the CAG promoter in pCAG-ZZEF1-ALOX15-T2A-L2T, generated using Geneious Prime. CpG dinucleotides are indicated by black vertical bars. (B) Bar chart showing the aggregate 5mC methylation probabilities (modification score, %) of CpG dinucleotides across all copies of the CAG promoter present within the concatemer, indicating hypermethylation within intact copies of the transgene promoter. Methylation status was called from PacBio HiFi reads using Jasmine and analyzed using pb-CpG-tools. Colored bars denote sites within the CMV enhancer, chicken β-actin promoter, and chimeric intron regions. The horizontal dashed line indicates the 50% modification score threshold determining CpG dinucleotide methylation status.

Article Snippet: 5-methylcytosine (5mC) profiling was then performed using pb-CpG-tools v3.0.0 ( https://github.com/PacificBiosciences/pb-CpG-tools/ ), where CpG dinucleotides with modification scores ≥50 were classified as methylated.

Techniques: Generated, Methylation, Modification

Journal: Current Protocols

Article Title: A Facile Strategy for 3′‐Terminal Functionalization of DNA and RNA via On‐Column Conjugation with Integration into Tandem Oligonucleotide Synthesis

doi: 10.1002/cpz1.70374

Figure Lengend Snippet: ( A ) Previously developed sulfonyl‐based solid support strategy for the incorporation of amino modifiers ( S ‐CPG). In some implementations, S ‐CPG can also be conjugated to the solid support through a carbamate linkage. ( B ) This work focuses on the use of CPR II CPG to incorporate amino modifiers via initial activation with N , N ′‐disuccinimidyl carbonate (DSC), generating R ‐CPG. A trifunctional ligand (protected 1,3‐diamino‐2‐propanol) enables anchoring to the solid support, followed by selective conjugation at the amine and subsequent strand elongation from the hydroxyl group.

Article Snippet: Importantly, CPR II CPG is compatible with diethylamine treatment, which may be useful in removing electrophilic species capable of reacting with strongly nucleophilic amines (Glen Research, ).

Techniques: Activation Assay, Conjugation Assay

Journal: Current Protocols

Article Title: A Facile Strategy for 3′‐Terminal Functionalization of DNA and RNA via On‐Column Conjugation with Integration into Tandem Oligonucleotide Synthesis

doi: 10.1002/cpz1.70374

Figure Lengend Snippet: ( A ) Synthesis of the crude 5′‐ O ‐(4,4′‐dimethoxytrityl)‐3′‐ O ‐sucinimide thymidine ( 1 ) for use in solid support reactions. ( B ) Activation of R ‐CPG using DSC‐mediated mixed carbonate synthesis. ( C ) On‐solid‐support construction of a putrescine carbamate‐linked nucleoside, which can be used for strand elongation subsequently. Reagent abbreviations: DSC, N,N ′‐disuccinimidyl carbonate, CPR II CPG, chemical phosphorylation reagent II control pore glass. Note : All round‐bottom flasks should be oven‐dried at 110°C for at least 2 hr before DSC activation to ensure complete removal of residual moisture. In addition, 3‐Å or 4‐Å molecular sieves should be preactivated if long‐term storage, defined as 2‐3 days, of the crude carbonate solution is anticipated, as they help maintain anhydrous conditions and minimize hydrolysis of the mixed carbonate. An automated dispensing pipettor should be used for measuring liquid volumes, and a scoopula should be used for transferring solid reagents to ensure accuracy and reproducibility. The following reagents are needed to make a mixed DNA oligonucleotide: 3% (w/v) trichloroacetic acid in dichloromethane (DCM; ChemGenes, cat. no. RN‐1462); cap A (acetic anhydride/pyridine/tetrahydrofuran [THF]; ChemGenes, cat. no. RN‐1458); cap B (10% N ‐methylimidazole in THF; ChemGenes, cat. no. RN‐1481); activation reagent (0.25 M of 5‐ethylthio‐tetrazole in ACN; ChemGenes, cat. no. RN‐1466); oxidation solution (0.02 M iodine/pyridine/H 2 O/THF; ChemGenes, cat. no. RN‐1455); acetonitrile (ACN), DNA synthesis grade (Fisher Scientific, cat. no. A998‐4); 2′‐deoxyadenosine (n‐bz) CED phosphoramidite (ChemGenes, cat. no. ANP‐5551); 2′‐deoxyguanosine (n‐ibu) CED phosphoramidite (ChemGenes, cat. no. ANP‐5553); 2′‐deoxycytidine ( n ‐acetyl) CED phosphoramidite (ChemGenes, cat. no. ANP‐5560); and thymidine CED phosphoramidite (ChemGenes, cat. no. ANP‐5554).

Article Snippet: Importantly, CPR II CPG is compatible with diethylamine treatment, which may be useful in removing electrophilic species capable of reacting with strongly nucleophilic amines (Glen Research, ).

Techniques: Activation Assay, Phospho-proteomics, Control, Transferring, DNA Synthesis