Sequence detection software v2

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Sequence Detection Software V2.3 is a bioinformatics software tool developed by Thermo Fisher Scientific. The core function of the software is to analyze and interpret DNA sequence data generated from various sequencing platforms.

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Sequence Detection Software (33 citations)

10 protocols using sequence detection software v2

Validating PRPF31 Gene Deletions

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Validation of the large deletion in the gene PRPF31 was performed in the two affected and six unaffected members of family RP-0777 using quantitative PCR (qPCR) with two different methods: TaqMan assays using the predesigned probes Hs01877341_cn (chr19:54,618,875) and Hs01993463_cn (chr19:54,619,056) (Applied Biosystems TaqMan Copy Number Assays, Life Technologies, Inc.) and the Universal Probe Library (UPL; Roche, Indianapolis, IN) with slight modifications of what was previously described in [28 (link)] and [29 (link)]. Briefly, primer and UPL probe combinations were designed against PRPF31 genomic DNA sequence using the Probe Finder v2.49 software (Roche, Indianapolis, IN). Five assays spanning the length of the gene were selected for validation (genomic coordinates of each targeted amplicon listed in Table 1).
Quantitative PCR was performed on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Data were evaluated using the Sequence Detection Software v2.4 (Applied Biosystems, Foster City, CA) and further analyzed by the ΔΔCT method. The geometric mean of the CT values for the three control sequences (GAPDH, RPPH1, and SNCA) was calculated and used as the reference value for ΔCT calculations. Hemizygous deletions were determined when the relative copy number value for a specific sample normalized to the reference sample was less than 0.75.

Almoguera B., Li J., Fernandez-San Jose P., Liu Y., March M., Pellegrino R., Golhar R., Corton M., Blanco-Kelly F., López-Molina M.I., García-Sandoval B., Guo Y., Tian L., Liu X., Guan L., Zhang J., Keating B., Xu X., Hakonarson H, & Ayuso C. (2015). Application of Whole Exome Sequencing in Six Families with an Initial Diagnosis of Autosomal Dominant Retinitis Pigmentosa: Lessons Learned. PLoS ONE, 10(7), e0133624.

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Validating CNV Locus by qPCR

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Five available DNA samples from AN were used for the validation of the CNV locus using quantitative polymerase chain reaction (qPCR). Probes and primer sequences were selected using the ProbeFinder v2.49 software (Roche, Indianapolis, IN) from the Universal Probe Library (UPL, Roche, Indianapolis, IN). The probe and primers used in this study are: the probe #27, cat.no. 04687582001; the left primer sequence 5’-ctcaatcgagcacaactactgc-3’; the right primer Sequence 5’-gagtcgagagtttccattcca-3’; Amplicon Sequence 5’-ctcaatcgagcacaactactgcttcgacaggcagcttactggaatggaaactctcgactc-3’. The probe is in the NIPA2 gene. The qPCR was performed on an ABI Prism™ 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Each sample reaction was done in triplicate, in 10 ul of reaction mixture containing 10 ng genomic DNA, 100 nM of the UPL probe, 400 nM of each PCR primer, and 1× TaqMan Gene Expression Master Mix containing UDG and ROX (Life Technologies, Carlsbad, CA). Two samples of reference genomic DNA with normal copy number (Promega, Madison, WI) were included in the assay as normal controls. Data were collected using the Sequence Detection Software v2.4 (Applied Biosystems, Foster City, CA), and analyzed by the ∆∆CT method.

Chang X., qu H., Liu Y., Glessner J., Hou C., Wang F., Li J., Sleiman P, & Hakonarson H. (2019). Microduplications at the 15q11.2 BP1–BP2 locus are enriched in patients with anorexia nervosa. Journal of psychiatric research, 113, 34-38.

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Adiponectin mRNA Expression in Adipose Tissue

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Total RNA was extracted from the adipose tissue specimen with the miRNeasy Mini Kit (Qiagen). Reverse transcription was performed with the Invitrogen Complementary DNA Synthesis Kit and SuperScript IV VILO Master Mix (both from ThermoFisher Scientific) according to the manufacturers’ instructions. Adiponectin mRNA expression in the adipose tissue was measured by real-time quantitative polymerase chain reaction using the Applied Biosystems Step One Plus system. Commercially available TaqMan primers and probes, including 2 unlabeled polymerase chain reaction primers and 1 fluorescein amidite dye-labeled TaqMan minor groove label probe, were used (all from Applied Biosystems). Phosphoglycerate kinase-1 was used as the housekeeping gene because of its stable expression in human adipose tissue.²⁰ (link) The expression of adiponectin mRNA was compared to that of the adipose tissue from 6 individuals with no kidney disease. All samples were performed in triplicate. The results were analyzed with the Sequence Detection Software v2.0 (Applied Biosystems). The ΔΔCT method for relative quantitation was used.²¹ (link)

Than W.H., Chan G.C., Kwan B.C., Lai K.B., Chan R.C., Teoh J.Y., Ng J.K., Fung W.W., Chow K.M., Cheng P.M., Law M.C., Li P.K, & Szeto C.C. (2022). Circulating and Adipose Tissue Adiponectin Level and Outcomes in Incident Peritoneal Dialysis Patients. Kidney Medicine, 5(3), 100589.

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Quantitative Analysis of ZAG mRNA and Protein in Adipose Tissue

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The methods of RNA extraction have been described previously²⁰. ZAG mRNA expression in the adipose tissue was measured by real-time quantitative polymerase chain reaction (RT-QPCR), using the Applied Biosystems Step One Plus system (Foster City, CA). Commercially available Taqman primers and probes, including 2 unlabeled PCR primers and 1 FAM™ dye labeled TaqMan® MGB probe, were used (all from Applied Biosystems). We used the phosphoglycerate kinase-1 (Applied Biosystems) as the housekeeping gene. Results were analyzed with Sequence Detection Software v2.0 (Applied Biosystems) and the relative quantification method by ∆∆Ct was applied for expression of targets in fold compared to the expression detected in samples from healthy subjects.
Serum ZAG level was measured by the commercially available ELISA kit (Alpha-2 Glycoprotein/Glycoprotein/ZAG/A2GP1 Human ELISA kit, BMS 2201, Invitrogen™, Carlsbad, CA) following the instructions from manufacturer. All assays were performed in duplicate. The detection limit of ZAG is 0.174 ng/ml.

Chan G.C., Than W.H., Kwan B.C., Lai K.B., Chan R.C., Teoh J.Y., Ng J.K., Chow K.M., Fung W.W., Cheng P.M., Law M.C., Leung C.B., Li P.K, & Szeto C.C. (2022). Adipose and serum zinc alpha-2-glycoprotein (ZAG) expressions predict longitudinal change of adiposity, wasting and predict survival in dialysis patients. Scientific Reports, 12, 9087.

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Omentin-1 mRNA Expression in Adipose Tissue

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All adipose tissue specimens were processed immediately and stored at -80°C. Total RNA was extracted from the adipose tissue specimen using the miRNeasy Mini Kit (Qiagen). Reverse transcription was performed using the Invitrogen cDNA Synthesis Kit and SuperScript IV VILO Master Mix (both from ThermoFisher Scientific) according to the manufacturer’s instructions. Omentin-1 mRNA expression in the adipose tissue was measured by real-time quantitative polymerase chain reaction, using the Applied Biosystems Step One Plus system. Commercially available TaqMan primers and probes, including 2 unlabeled polymerase chain reaction primers and 1 fluorescein amidites dye-labeled TaqMan minor groove binder probe, were used (all from Applied Biosystems). Phosphoglycerate kinase-1 was used as the housekeeping gene because of its stable expression in human adipose tissue.²² (link) The expression of omentin-1 mRNA was compared to that of the adipose tissue of 6 individuals with no kidney disease. All samples were performed in triplicate. The results were analyzed with the Sequence Detection Software v2.0 (Applied Biosystems). The ΔΔCT method for relative quantitation was used.²³ (link)

Than W.H., Chan G.C., Kwan B.C., Lai K.B., Chan R.C., Teoh J.Y., Ng J.K., Fung W.W., Chow K.M., Cheng P.M., Li P.K, & Szeto C.C. (2023). Omentin-1 Levels and Outcomes in Incident Peritoneal Dialysis Patients. Kidney Medicine, 5(3), 100598.

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Real-time RT-PCR Analysis of Gene Expression

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Total RNA was converted into cDNA with High-Capacity cDNA RT Kit (Applied Biosystems). Real-time RT PCR was performed in triplicate for GAPDH, COX-2 (PTGS2), TSG-6 (TNFAIP6), TRAIL, IL-24, IL1A, and IL1B using Taqman® Gene Expression Assays (Life Technologies) and Taqman® Fast Master Mix (Life Technologies). Total of 10–20 ng of cDNA was used for each 20 µl reaction. Thermal cycling was performed with 7900HT System (Applied Biosystems) by incubating the reactions at 95°C for 20 s followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. Data were analyzed with Sequence Detection Software V2.3 (Applied Biosystems) and relative quantities (RQs) were calculated with comparative critical threshold (C_T) method using RQ Manager V1.2 (Applied Biosystems).

Ylostalo J.H., Bartosh T.J., Tiblow A, & Prockop D.J. (2014). Unique characteristics of human mesenchymal stem/progenitor cells (MSC) pre-activated in 3D cultures under different conditions. Cytotherapy, 16(11), 1486-1500.

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Production and Quantification of HPV16 Pseudovirions

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HPV16 PsVs were prepared as previously described^{63 (link)}. In brief, expression plasmids carrying codon-optimized L1 and L2^{64 (link)} expression vector pShell 16L1/L2wt^{65 (link)} were co-transfected with a pcDNA3.1 luciferase reporter plasmid^{66 (link)} into HEK 293TT cells using polyethylenimine. For PsVs used in infection assays, the pcDNA3.1 luciferase reporter plasmid was replaced by the promoter-reporter plasmid pGL4.20 containing the HPV16 long control region (LCR) and the HPV16 early promoter regulating the luciferase expression as described earlier^{67 (link),68 (link)}. For detection of the DNA, EdU-modified PsVs were used. After transfection of pShell 16L1/L2wt and pcDNA3.1 plasmids the cell culture medium was supplemented with 20 μM 5-ethynyl-2′-deoxyuridine (EdU, Click-iT AlexaFluor® 488 Imaging Kit, Thermo Fisher Scientific), to enable the staining of the DNA.
48 hours after transfection, cells were lysed and the pseudoviruses were purified by gradient centrifugation using OptiPrep (Sigma-Aldrich). Quantification of the pseudovirions (viral genome equivalents (vge)) was performed by quantitative PCR using a 7500 Real-Time PCR System and Sequence Detection Software v2.3 (Applied Biosystems, Foster City, CA, USA)^{69 (link)}.

Finke J., Mikuličić S., Loster A.L., Gawlitza A., Florin L, & Lang T. (2020). Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6. Scientific Reports, 10, 5356.

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Quantification of Gene Expression in Cell and Tissue Samples

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Total RNA was isolated from U937 cells or homogenized heart tissue using RNeasy Mini Kit (Qiagen, Valencia, CA) with DNase I (RNase-Free DNase Set, Qiagen) digestion step. The isolated RNA was quantified with nanodrop spectrophotometer (ThermoFisher Scientific, Rockford, IL) and converted to complementary DNA (cDNA) with High-Capacity cDNA RT Kit (Applied Biosystems, Technologies, Grand Island, NY). Real-time RT-PCR was performed in triplicate for expression of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), CD11b (ITGAM), CD14, TLR2, and TLR4, as well as mouse Tnf, Ucp2, and Ucp3 using TaqMan^® Gene Expression Assays (Applied Biosystems, Technologies) and TaqMan^® Fast Master Mix (Life Technologies). Total of 20 ng of cDNA was used for each 20 μl reaction. Thermal cycling was performed with 7900HT System (Applied Biosystems, Technologies) by incubating the reactions at 95° C for 20 seconds followed by 40 cycles of 95° C for 1 second and 60° C for 20 seconds. Data were analyzed with Sequence Detection Software V2.3 (Applied Biosystems, Technologies) and relative quantitation (RQ) was calculated with comparative critical threshold (Ct) method using RQ Manager V1.2 (Applied Biosystems, Life Technologies).

Mohammadipoor A., Lee R.H., Prockop D.J, & Bartosh T.J. (2016). Stanniocalcin-1 Attenuates Ischemic Cardiac Injury and Response of Differentiating Monocytes/Macrophages to Inflammatory Stimuli. Translational research : the journal of laboratory and clinical medicine, 177, 127-142.

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Genotyping Key Addiction-Related SNPs

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SNPs rs1799971 (A118G) in OPRM1, rs910080 in PDYN and rs6473797 in OPRK1 were genotyped using pre-designed TaqMan^® SNP Genotyping Assays and Universal PCR master mix (all from Applied Biosystems, Foster City, CA) in a 384-well plate. Polymerase chain reaction (PCR) cycling was done on an ABI Prism^® 9700 Thermocycler (Applied Biosystems, Foster City, CA) and the endpoint analysis was performed using an ABI Prism^® 7900HT Sequence Detection System using Sequence Detection Software v.2.3 (Applied Biosystems, Foster City, CA).

Randesi M., Rotrosen J., Nunes E.V., Lee J.D., Novo P., Levran O., Ott J., Pavlicova M., Scodes J, & Jeanne Kreek M. (2020). Variants of opioid genes and response to treatment of opioid use disorder with buprenorphine-naloxone versus extended-release naltrexone in Caucasians. The American journal of drug and alcohol abuse, 46(6), 761-768.

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Gene Expression Analysis of Cultured Arteries

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Total RNA was obtained from cultured arteries or cells and cDNA was obtained by reverse-transcription (see online supplementary methods). Specific pre-developed TaqMan probes from Applied Biosystems (TaqMan Gene Expression Assays) (see online supplementary table S3) were used for PCR amplification. Fluorescence was detected with ABI PRISM 7900 Hardware and results were analysed with the Sequence Detection Software V.2.3 (Applied Biosystems). Gene expression was normalised to the expression of the endogenous control GUSb using comparative ΔCt method. 5-7 11 mRNA concentration was expressed in relative units with respect to GUSb expression.

Corbera-Bellalta M., Planas-Rigol E., Lozano E., Terrades-García N., Alba M.A., Prieto-González S., García-Martínez A., Albero R., Enjuanes A., Espígol-Frigolé G., Hernández-Rodríguez J., Roux-Lombard P., Ferlin W.G., Dayer J.M., Kosco-Vilbois M.H, & Cid M.C. (2016). Blocking interferon γ reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis. Annals of the rheumatic diseases, 75(6).

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