Lipid nanoparticle-messenger RNA (LNP-mRNA) drug products are a growing class of drug modalities. The unique composition of these drug products requires multiple measurements to account for the different components of these drug modalities. Pharmacokinetic (PK) measurements include measurement of the encapsulated mRNA and components of the LNP in circulation to understand the effectiveness of the therapeutic mRNA. The PK measurements can utilize many different platforms including PCR. Current regulatory guidance documents for bioanalytical method validation are specific to ligand binding and chromatographic assay methods and difficult to interpret for use with molecular workflows. The purpose of this paper is to provide information on considerations for validation of regulated reverse transcription quantitative PCR (RT-qPCR) assays that are used to support the pharmacokinetic analysis of LNP-mRNA drug products.

• Klíčová slova
• LNP, MRNA, PK, RT-dPCR, RT-qPCR,
• MeSH
• kvantitativní polymerázová řetězová reakce * metody   MeSH
• lidé MeSH
• lipidy * chemie   farmakokinetika   MeSH
• liposomy MeSH
• messenger RNA * farmakokinetika   aplikace a dávkování   MeSH
• nanočástice * chemie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Lipid Nanoparticles MeSH Prohlížeč
• lipidy * MeSH
• liposomy MeSH
• messenger RNA * MeSH

The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.

• Klíčová slova
• AAV, RT-qPCR, biodistribution, cell therapy, cellular kinetics, dPCR, gene therapy, qPCR, shedding, transgene expression,
• MeSH
• genetická terapie * MeSH
• polymerázová řetězová reakce MeSH
• tkáňová distribuce MeSH
• vyvíjení léků * MeSH
• Publikační typ
• časopisecké články MeSH

DNA origami nanoframes with two parallel DNA sequences are used to evaluate the effect of nucleoside substituents on radiation-induced DNA damage. Double strand breaks (DSB) of DNA are counted using atomic force microscopy (AFM), and total number of lesions is evaluated using real-time polymerase chain reaction (RT-PCR). Enhanced AT or GC content does not increase the number of DNA strand breaks. Incorporation of 8-bromoadenosine results in the highest enhancement in total number of lesions; however, the highest enhancement in DSB is observed for 2'-deoxy-2'-fluorocytidine, indicating different mechanisms of radiosensitization by nucleoside analogues with the halogen substituent on base or sugar moieties, respectively. "Bystander" effects are observed, when the number of DSB in a sequence is enhanced by a substituent in the parallel DNA sequence. The present approach eliminates limitations of previously developed methods and motivates detailed studies of poorly understood conformation or bystander effects in radiation induced damage to DNA.

• MeSH
• adenosin analogy a deriváty   MeSH
• deoxycytidin analogy a deriváty   MeSH
• DNA * účinky záření   MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 2'-fluoro-2'-deoxycytidine MeSH Prohlížeč
• 8-bromoadenosine MeSH Prohlížeč
• adenosin MeSH
• deoxycytidin MeSH
• DNA * MeSH

The identification of sex in larvae of insects is usually challenging or even impossible, while in adults the sexual dimorphism is usually evident. Here, we used copy number analysis to develop a method of sex detection in Colorado potato beetle (Leptinotarsa decemlineata), which has an X0 sex determination system. The X linked gene LdVssc and autosomal gene LdUBE3B were identified as appropriate target and reference loci, respectively. The copy numbers (CNV) of LdVssc in males and females were estimated using standard droplet digital PCR (ddPCR) and real-time PCR (qPCR). With both methods, CNVs were bimodally distributed (BAddPCR = 0.709 and BAqPCR = 0.683) with 100% ability to distinguish females from males. The use of qPCR-based sex detection in a broad collection of 448 random CPB adults showed a perfect association (Phi = 1.0, p < 0.05) with the true sexes of adults, with mean CNV in females of 2.032 (SD = 0.227) and 0.989 in males (SD = 0.147). In the collection of 50 random 4th instar larvae, 27 females and 23 males were identified, consistent with the expected 1:1 sex ratio (p = 0.689). The method is suitable for sexing in all stages of ontogenesis. The optimal cost-effective application of the method in large populations requires the DNA extraction using CTAB, the qPCR assay in one biological replicate and three technical replicates of each marker, and the use of one randomly chosen male per run to calibrate calculation of CNV.

• MeSH
• brouci * genetika   MeSH
• larva genetika   MeSH
• Solanum tuberosum * MeSH
• variabilita počtu kopií segmentů DNA MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Colorado MeSH

Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.

• Klíčová slova
• RT-qPCR, gene expression, preamplification, quantitative PCR, reverse transcription, sample collection, single cell,
• MeSH
• analýza jednotlivých buněk metody   normy   MeSH
• kvantitativní polymerázová řetězová reakce metody   normy   MeSH
• lidé MeSH
• reverzní transkripce genetika   MeSH
• senzitivita a specificita MeSH
• stanovení celkové genové exprese metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH