Repli g mitochondrial dna kit

Genomic DNA was extracted from the purified samples using a commercial kit (QIAamp DNA Mini Kit, Qiagen, Germany); then, mtDNA was amplified using the REPLI-g Mitochondrial DNA Kit (Qiagen, Germany). Using the Nanodrop spectrophotometer ND-2000c (Thermo Scientific, USA), only the isolated DNA with an optimal density ratio of 260/280 of 1.8 or more was chosen and stored at − 20 °C.

All probands from families (n = 77) with familial forms of diabetes were screened for mutations in six known MODY genes (i.e. those accounting for the more prevalent forms of MODY) [6 (link)]. In addition, also the presence of the A3243G mitochondrial mutation, responsible of the maternally inherited diabetes and deafness (MIDD) was assessed.
Total DNA was extracted from peripheral blood according to standard procedures.
All exons, flanking introns, and minimal promoter regions of HNF4A, GCK, HNF1A, PDX1, HNF1B, and NeuroD1 were amplified from a genomic DNA sample of each proband by polymerase chain reaction (PCR), using gene-specific oligonucleotide primers (S1 Table). Amplicons were subjected to direct sequencing on an automated ABI 3100 (Applied Biosystems, CA), using the ABI Prism BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, CA). Results were analysed with Sequencher software v5.0 (GeneCodes, MI).
Mitochondrial DNA fraction was enriched from total DNA sample of each subject by using the REPLI-g Mitochondrial DNA Kit (Qiagen) following manufacturer’s protocol.
The presence of the A3243G mitochondrial mutation was assessed by PCR and direct sequencing using mitochondrial genome-specific oligonucleotide primers as previously reported [7 (link)].

Mitochondrial consensus sequences were established for every transplant donor and recipient. DNA was extracted from whole blood samples (Qiagen DNeasy Blood & Tissue kit) collected pre-transplant. Mitochondrial DNA was selectively amplified (Qiagen REPLI-g Mitochondrial DNA Kit), and sheared to 300 bp (Covaris). Libraries were prepared for sequencing using the NEBNext Ultra library preparation, characterized (Advanced Analytical Fragment Analyzer and dPCR) and sequenced (2 × 250 bp, Illumina MiSeq). One million sequences led to a per-base coverage greater than 100-fold (genome size 16.5 kb), sufficient to determine subject-specific mitochondrial variants. Fastq files were trimmed (Trimmomatic34 (link), LEADING:25 TRAILING:25 SLIDINGWINDOW:4:30 MINLEN:15) and aligned against the human reference genome [GenBank:GCA_000001305.2] using BWA-mem35 (link). Sequences that mapped to the mitochondrial reference sequence (edited from [GenBank:NC_012920) were extracted. A BCF file of SNPs was created and a FASTA consensus sequence was determined. A list of informative SNPs was created.

Total DNA was extracted from either FFPE adipose tissue sections of MD patients (n = 8) or ASC at the third passage in culture (n = 3 ASC and n = 3 MD-ASC) using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s recommendations. The extracted DNA concentration was determined by a spectrophotometer measurement (Nanodrop2000 Thermo Scientific™, Waltham, MA, USA) from the absorbance at 260 nm.
To separate mtDNA from linear DNA molecules, a mitochondrial specific amplification was carried out with the REPLI-g Mitochondrial DNA Kit (Qiagen, Hilden, Germany) loading 50 ng of total DNA template per 50 μL of final reaction buffer. The kit enables selective amplification of mitochondrial DNA from total DNA samples without the need for prior mitochondrial DNA isolation.