CDKN3 RNA variants were determined in the cDNA of 45 tumors, 22 normal cervical epithelium, and three cell lines (CaSki, Hela, and SiHA) using RT-PCR. The primers used for the RT-PCR were previously described [27 (link)] or designed according to published variants sequences [28 (link)] (
Abi prism 3130xl genetic analyzer system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM 3130xl Genetic Analyzer is a capillary electrophoresis-based DNA sequencing system. It features 16 capillaries and can perform high-throughput DNA analysis. The system is designed for a range of genetic applications, including DNA sequencing, fragment analysis, and genotyping.
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Lab products found in correlation
Bigdye terminator ready reaction kit, by Thermo Fisher Scientific (3 mentions)
Big dye terminator version 3.1 kit, by Thermo Fisher Scientific (2 mentions)
Seqscape software, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Bigdye terminator ready reaction kit, by PerkinElmer (1 mentions)
Clc bio main workbench, by Qiagen (1 mentions)
Pgem t easy plasmid, by Promega (1 mentions)
Qiaquick spin kit, by Qiagen (1 mentions)
Ptc 100 peltier thermal cycler, by Bio-Rad (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
8 protocols using abi prism 3130xl genetic analyzer system
1
Characterizing CDKN3 RNA Variants in Cancers
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CDKN3 RNA variants were determined in the cDNA of 45 tumors, 22 normal cervical epithelium, and three cell lines (CaSki, Hela, and SiHA) using RT-PCR. The primers used for the RT-PCR were previously described [27 (link)] or designed according to published variants sequences [28 (link)] (S1 Table ). The PCR products were analyzed using agarose gel electrophoresis and sequenced using the fluorescent cycle-sequencing method (BigDye Terminator Ready Reaction Kit; Applied Biosystems). Sequence analysis was performed using an ABI PRISM 3130xl Genetic Analyzer system (Applied Biosystems). Sequences were analyzed with the FASTA sequence similarity tool [13 , 26 (link)], SeqScape software (Applied Biosystems), and ClustalW2 alignment tool (http://www.ebi.ac.uk/Tools/msa/clustalw2/ ).
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CDKN3 RNA variants were determined in the cDNA of 45 tumors, 22 normal cervical epithelium, and three cell lines (CaSki, Hela, and SiHA) using RT-PCR. The primers used for the RT-PCR were previously described [27 (link)] or designed according to published variants sequences [28 (link)] (
2
HPV Detection by PCR and Sequencing
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The HPV detection was performed by PCR using universal primers located in HPV L1 genes MY09/MY11, GP5+/6+, and L1C1, as described previously [23 (link)–25 (link)]. The HBB gene was used as an internal control to assess the quality of the DNA. The HPV types were identified by sequencing the amplified bands using the fluorescent cycle-sequencing method (BigDye Terminator Ready Reaction Kit; Applied Biosystems, Carlsbad, CA, USA). Sequence analysis was performed using an ABI PRISM 3130xl Genetic Analyzer system (Applied Biosystems). Each band sequenced was analyzed with the FASTA sequence similarity tool [13 , 26 (link)]. The average identity percentage of HPV types detected was 98.7% when compared to the reference sequences.
3
HPV Detection by PCR and Sequencing
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HPV detection was performed by PCR using universal primers located in the HPV L1 gene (MY09/MY11, GP5+/6+, and L1C1), as described previously.19 The HBB gene was used as an internal control to assess the quality of the DNA. The HPV types were identified by sequencing the amplified bands using the fluorescent cycle‐sequencing method (BigDye Terminator Ready Reaction Kit; Applied Biosystems, Carlsbad, CA, USA). Sequence analysis was performed using an ABI PRISM 3130xl Genetic Analyzer system (Applied Biosystems). Each band sequenced was analyzed with the FASTA sequence similarity. The average identity percentage of HPV types detected was 98.7% (91–100%) when compared to the reference sequences.
4
Molecular Identification of Aspergillus Isolates
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The species identification of two isolates (an aflatoxigenic isolate MEX-A19-13 and an atoxigenic isolate MEX-A19-2nd-5) was conducted at Techno Suruga Laboratory (Shizuoka, Japan). Genomic DNA was extracted, and the genome sequences of the cmd genes were analyzed, followed by PCR amplification with CMD5 and CMD6 primers [32 (link)]. The resulting PCR products were sequenced using an ABI PRISM 3130xl Genetic Analyzer System (Applied Biosystems, Foster City, CA, USA). The obtained sequence data ere compared with those from DNA Data Bank of Japan (DDBJ) by using the BLAST search. The phylogenetic tree analysis was conducted using Aporon 3.0 (Techno-Suruga Lab., Shizuoka, Japan), on the basis of the neighbor-joining method of cmd gene sequence data.
The genomic nucleotide sequence data for the cmd genes of MEX-A19-13 and MEX-A19-2nd-5 were deposited in the DDBJ/ European Molecular Biology Laboratory (EMBL)/GenBank nucleotide sequence database under the accession nos. LC383381 and LC383382.
The genomic nucleotide sequence data for the cmd genes of MEX-A19-13 and MEX-A19-2nd-5 were deposited in the DDBJ/ European Molecular Biology Laboratory (EMBL)/GenBank nucleotide sequence database under the accession nos. LC383381 and LC383382.
5
Profiling Bacteroides UGL Genes
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Specific primers of the Bacteroides UGL gene were designed based on the nucleotide sequences of genes coding for five Bacteroides UGLs (Bache_1615 of B. helcogenes P 36–108, BT3348 of B. thetaiotaomicron VPI-5482, BXY_37650 of Bacteroides xylanisolvens XBIA, BF0331 of Bacteroides fragilis NCTC 9343 and BVU_0115 of B. vulgatus ATCC 8482) (Table S2 ). RT-PCR was conducted in the TechnoSuruga Laboratory using total DNA extracted from the faeces of the two men in their 20’s and 40’s as a template and primers for 16S rRNA or the Bacteroides UGL gene with Illumina HiSeq T. The primers (341f and 534r) for the 16S rRNA gene are shown in Table S2 . In the same way, RT-PCR was performed using genomic DNA from B. vulgatus as a positive control. The gene fragments amplified by RT-PCR were subcloned to a pGEM-T Easy Vector System (Promega) and each of clones from the two men was subjected to a determination of nucleotide sequence by the ABI PRISM 3130xl Genetic Analyzer System (Applied Biosystems), as previously described33 (link).
6
Cloning and Phylogenetic Analysis of RACE Sequences
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Cloning and sequence analysis. The RACE product bands were excised from the agarose gels and purified using the QIAquick Spin kit (Qiagen) according to the manufacturer's protocol. After purification, the RACE products were cloned into the pGEM T-Easy plasmid (Promega). Plasmids from positive colonies were analyzed by restriction; each clone was sequenced in both chains using universal primers and the Big Dye Terminator Ready Reaction kit (Perkin-Elmer) and analyzed in the ABI PRISM 3130xl Genetic Analyzer System (Applied Biosystems). Sequence information was analyzed using the CLC Bio Main Workbench (CLC Bio; Qiagen) as well as the BLAT (genome.ucsc.edu) and BLAST (blast.ncbi. nlm.nih.gov) algorithms.
In order to search for sequence similarity among clones, we performed a CLUSTAL alignment and checked it manually. Based on this alignment, we searched for the most adequate model of evolution using the FindModel server (http://www. hiv.lanl.gov/content/sequence/findmodel/findmodel.html), and the GTR model was selected to perform a maximum likelihood phylogenetic analysis employing a 1000-replicate bootstrap analysis. Finally, a tree was constructed using the neighborjoining method.
In order to search for sequence similarity among clones, we performed a CLUSTAL alignment and checked it manually. Based on this alignment, we searched for the most adequate model of evolution using the FindModel server (http://www. hiv.lanl.gov/content/sequence/findmodel/findmodel.html), and the GTR model was selected to perform a maximum likelihood phylogenetic analysis employing a 1000-replicate bootstrap analysis. Finally, a tree was constructed using the neighborjoining method.
7
Genotyping of IL-10 SNPs in Blood
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Genomic DNA was isolated from 300 μL EDTA-treated whole blood using a Commercial kit (Wizard genomic DNA purification kit, Promega, WI, USA). The procedure was carried according to the kit manufacturer's recommendation.
Genotyping analysis for detection of 3 SNPs of IL-10s was performed for all patients by using specific PCR primers.Table 1 describes primers used and PCR conditions. PCR was performed in a total volume of 25 μL using 100 ng of genomic DNA with 1.5 μL of 10 μmol/L of each primer and 12.5 μL of 2X KAPA2G Fast ReadyMix PCR Kit (Kappa Biosystems, USA). PCR amplifications were performed in PTC-100 Peltier Thermal Cycler (MJ Research, MA, USA).
PCR reaction products were sequenced using Big Dye Terminator version 3.1 kit (Applied Biosystems, Waltham, MA, USA). Samples were run on an ABI Prism Genetic Analyzer system 3130xl (Applied Biosystems, Waltham, MA, USA).
Genotyping analysis for detection of 3 SNPs of IL-10s was performed for all patients by using specific PCR primers.
PCR reaction products were sequenced using Big Dye Terminator version 3.1 kit (Applied Biosystems, Waltham, MA, USA). Samples were run on an ABI Prism Genetic Analyzer system 3130xl (Applied Biosystems, Waltham, MA, USA).
8
Amplification and Sequencing of XRCC Genes
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The PCR amplifications targeting the XRCC1-exon-10, XRCC3-exon-7 and XRCC3-5’-UTR regions were performed using specific primers based on the XRCC1 and XRCC3 sequences obtained from the National Center for Biotechnology Information (NCBI) (Table 1
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