Flexigene dna kit

Genomic DNA was extracted from the monocytes in peripheral blood, with the QIAGEN FlexiGene DNA kit (Qiagen, Germany) following the standard protocol. DNA concentration was tested by a NanoDrop spectrophotometer. DNA samples were stored at −20°C before use.

1 mL of blood was collected in EDTA-coated vacutainer. Genomic DNA (gDNA) was extracted from the collected blood using QIAGEN flexigene® DNA kit (Qiagen, Hilden, Germany) following manufacturer’s instructions.

DNA was extracted from hESC lines following culture on matrigel (1:100 in KO-DMEM) using the QIAGEN-flexigene DNA kit (Cat # 51204, QIAGEN). DNA extraction was performed according to the manufacturer’s protocol. Amplification of hESCs DNA was performed encompassing the MCR region, the APC gene "hot spots" and the germline mutation. The PCR products were purified by the QIAquick PCR purification kit (QIAGEN) following the manufacturer's instructions, and sequenced using the ABI330XL (Sequencer ABI, Center of the Life Sciences Faculty in Tel Aviv University).
The entire APC gene was also sequenced using Pronto Diagnostics kit specifically aimed at sequencing all the coding sequences of the APC gene. The extracted DNA was sent to ProntoLab™, Pronto Diagnostics' molecular services laboratory (Tel Aviv, Israel) which used Multiplicom's (Neil, Belgium) FAP MASTR™ and MID Dx 1–48 for Illumina MiSeq® kits for library preparation. Illumina’s (San Francisco, CA) MiSeq Reagent Kit v2 (500 cycle) was used to run the library on the MiSeq instrument. The FAP MASTR™ kit enables identification of point mutations by complete coverage of all coding sequences of the APC gene. Data analysis was carried out using SeqNext module v4.1.2 of the Sequence Pilot software (JSI medical systems, Kippenheim, Germany).

DNA was extracted from 500 μL of whole blood in 500 μL RNAlater (Thermo Fisher Scientific, Waltham, MA) that had been frozen at -20°C. Samples were centrifuged at 13,500 RPM for 2 minutes and RNAlater was decanted off. DNA extractions were done using the QIAGEN FlexiGene DNA Kit (QIAGEN, Hilden, Germany) and quantified using fluorometric quantitation through the Promega QuantiFluor dsDNA System (Promega, Madison, WI) and BioTek Synergy spectrometer (BioTek, Winooski, VT). Quality was assessed through electrophoresis on a 1% agarose gel. Extraction via phenol-chloroform, QIAGEN DNeasy Kit (QIAGEN, Hilden, Germany), and Roche DNA Isolation Kit for Mammalian Blood (Roche, Basel, Switzerland) were also tested; however, the FlexiGene DNA Kit yielded the best results for our samples, in terms of both quantity of quality. DNA was concentrated as needed to meet a minimum concentration of 50 ng/μL.

Genomic DNA (gDNA) was used for mutation detection in 5´ untranslated region (UTR), 3´ UTR and exon 9 to exon 13 in G6PD deficient specimens, whereas mRNA was used for mutation detection in exon 1 to exon 10. The rationale behind use of RNA was avoiding large-sized intron in between exons (especially exon 2 and exon 3) and to amplify 4–5 exons using one pair of primers at a time. On the other hand DNA instead of RNA were used to get full-length sequence of UTRs of two different transcript variants (variant-1 and variant-2) of G6PD gene for mutation detection because RNA specimens were not suitable for full-length UTR sequencing. gDNA from whole blood was extracted according to guidelines of the QIAGEN flexigene® DNA kit (QIAGEN, Hilden, Germany) manual. On the other hand, Trizol® LS-based RNA extraction from whole blood was performed according to manufacturer’s instructions (Life Technologies, CA, USA).

DNA was extracted from whole blood using the QIAgen FlexiGene DNA Kit (Qiagen, Hilden, Germany). All DNA samples were subjected to the Sanger sequencing of coding regions and flanking intron regions of the APC gene as described earlier [12 (link)] and according to the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA). Allelic variants were confirmed by sequencing both DNA strands on an independent PCR product. Reactions were analyzed using the Applied Biosystems genetic analyzer ABI3130 (Life Technologies, Carlsbad, CA, USA). Large deletions or duplications of the APC gene were not analyzed. For the annotation of APC variants the following databases were used: INSIGHT-group database (http://www.insight-group.org/mutations/), National Center for Biotechnology Information (NCBI) SNP database, LOVD database, The Universal Mutation Database (UMD-APC), NCBI ClinVar database, and in-silico prediction tools MutationTaster and Mutalyzer [7 (link),13 (link),14 (link),15 (link),16 (link)]. Interpretation of the germline variants was made according to the “Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology” (2015) [17 (link)].