Genotyping of single nucleotide polymorphisms using the SNP-RFLP method

Saifullah, Saifullah; Tsukahara, Toshifumi

doi:10.5582/bst.2018.01102

Cited by 12 publications

(8 citation statements)

References 18 publications

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“…Sanger sequencing was performed using a 3130XL Genetic Analyzer (Applied Biosystems, Japan) as described in our previous protocol [34]. In some cases, 500-800 ng samples of plasmids with specific primers (Table S3) were sent to a company (Eurofins Genomics) for DNA sequencing.…”

Section: Sanger Sequencingmentioning

confidence: 99%

Effective RNA Knockdown Using CRISPR-Cas13a and Molecular Targeting of the EML4-ALK Transcript in H3122 Lung Cancer Cells

Saifullah

Sakari

Suzuki

et al. 2020

IJMS

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RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA–guide RNA complex residing at the 5′ of the crRNA. The activity of Cas13a was maximal for guide RNAs 24–30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70–76%), mCherry fluorescence (72%), and EML4-ALK at the protein (>80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.

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Section: Sanger Sequencingmentioning

confidence: 99%

Effective RNA Knockdown Using CRISPR-Cas13a and Molecular Targeting of the EML4-ALK Transcript in H3122 Lung Cancer Cells

Saifullah

Sakari

Suzuki

et al. 2020

IJMS

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“…The primers and sequences for CYP3A5*3 are as follows: forward primers (5'-CCTGCCTTCAATTTTCACT-3'); reverse primers (5'-GGTCCAAACAGGGAAGAGGT-3'). To validate the RT-PCR results, CYP3A5*3 (rs776746) was confirmed via Sanger sequencing using a 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, United States) ( Saifullah and Tsukahara, 2018 ; Allegri et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

CYP3A5 Genotype-Dependent Drug-Drug Interaction Between Tacrolimus and Nifedipine in Chinese Renal Transplant Patients

et al. 2021

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Purpose: The drug-drug interactions (DDIs) of tacrolimus greatly contributed to pharmacokinetic variability. Nifedipine, frequently prescribed for hypertension, is a competitive CYP3A5 inhibitor which can inhibit tacrolimus metabolism. The objective of this study was to investigate whether CYP3A5 genotype could influence tacrolimus-nifedipine DDI in Chinese renal transplant patients.Method: All renal transplant patients were divided into CYP3A5*3/*3 homozygotes (group I) and CYP3A5*1 allele carriers (CYP3A5*1/*1 + CYP3A5*1/*3) (group II). Each group was subdivided into patients taking tacrolimus co-administered with nifedipine (CONF) and that administrated with tacrolimus alone (Controls). Tacrolimus trough concentrations (C0) were measured using high performance liquid chromatography. A retrospective analysis compared tacrolimus dose (D)-corrected trough concentrations (C0) (C0/D) between CONF and Controls in group I and II, respectively. At the same time, a multivariate line regression analysis was made to evaluate the effect of variates on C0/D.Results: In this study, a significant DDI between tacrolimus and nifedipine with respect to the CYP3A5*3 polymorphism was confirmed. In group I (n = 43), the C0/D of CONF was significantly higher than in Controls [225.2 ± 66.3 vs. 155.1 ± 34.6 ng/ml/(mg/kg); p = 0.002]. However, this difference was not detected in group II (n = 27) (p = 0.216). The co-administrated nifedipine and CYP3A5*3/*3 homozygotes significantly increased tacrolimus concentrations in multivariate line regression analysis.Discussion: A CYP3A5 genotype-dependent DDI was found between tacrolimus and nifedipine. Therefore, personalized therapy accounting for CYP3A5 genotype detection as well as therapeutic drug monitoring are necessary for renal transplant patients when treating with tacrolimus and nifedipine.

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“…One potential modality would be the addition of a short myofiber-specific nuclear signal peptide (of human origin) to the peptide-conjugated PMO, which theoretically could enhance the nuclear internalization of target ASOs. Since genes associated with drug absorption, dispensation, metabolism, and clearance are polymorphic, efficacy and toxicity of drugs vary among individuals ( Jittikoon et al, 2016 ; Saifullah and Tsukahara, 2018 ; Saifullah, 2018 ; Saifullah et al, 2019 ). Therefore, personalized genetic medicine could enhance drug responses in DMD patients.…”

Section: Introductionmentioning

confidence: 99%