Sequence detection software version 2

Manufactured by Thermo Fisher Scientific

Sourced in United States

Sequence Detection Software version 2.0 is a software tool for analyzing and interpreting data from real-time PCR (polymerase chain reaction) experiments. The software provides functionality for designing, running, and analyzing real-time PCR experiments.

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11 protocols using sequence detection software version 2

DNA Isolation and ANKK1 Genotyping

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After the acquisition of clinical items a blood sample was taken. Isolation of DNA form was performed using the QIAamp DNA Blood Mini Kit (# 51106, QIAGEN) according to the manufacturer's instructions. Concentration and quality of the DNA were determined with an ND-1000 UV/Vis- Spectrophotometer (Peqlab). SNP genotyping for rs1800497 (ANKK1) was performed with 20 ng of DNA in triplicates using allelic discrimination assays (TaqMan SNP Genotyping Assays, Applied Biosystems by Invitrogen). The genotyping PCR was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems), and the resulting fluorescence data were analysed with Sequence Detection Software version 2.3 (Applied Biosystems). Subgroups were defined according to the gene variant defined by the ANKK1 TaqIA (rs1800497) SNP [A1- (wild type): GG; A1+ (risk allele): AG, AA].

Pelzer E.A., Melzer C., Schönberger A., Hess M., Timmermann L., Eggers C, & Tittgemeyer M. (2019). Axonal degeneration in Parkinson's disease – Basal ganglia circuitry and D2 receptor availability. NeuroImage : Clinical, 23, 101906.

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Quantifying miR-15b Expression via RT-qPCR

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RT-qPCR was conducted using TaqMan^® MicroRNA RT Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). miR-24 was used as an internal control [stem-loop RT primer sequence: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTTCTG-3', forward primer: 5'-GCGGCGGTGGCTCAGTACAGC-3' for miR-24, and (universal) reverse primer: 5'-GTGCAGGGTCCGAGGT-3'] during the quantification using specific stem-loop primers for miR-15b (stem-loop RT primer sequence: 5'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTGTAAACC-3'; forward primer: 5'-ATCCAGTGCGTGTCGTG-3', and reverse primer: 5'-TGCTTAGCAGCACATCATG-3') to normalize it as follows: ∆Ct=∆CtmiR-∆CtmiR-24(24 (link)). The analysis of the results was performed using Sequence Detection Software version 2.3 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The difference in the miR-15b relative expression levels between samples was calculated using the 2^-ΔΔCq method (25 (link)).

Mohany K.M., Al Rugaie O., Al-Wutayd O., Alsharidah M, & Al-Nafeesah A. (2022). Circulating miR-15b, Annexin A1, procalcitonin and interleukin-6 levels differentiate children with metabolically unhealthy obesity from those with metabolically healthy obesity: A case-control study. Experimental and Therapeutic Medicine, 23(6), 403.

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SNP Genotyping via TaqMan Assays

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Allele discrimination of SNP variants was performed using commercial TaqMan probes (Applied Biosystems, CA, United States) at a concentration of 20X. We selected three SNPs: rs13118928 (commercial probe id: C__11375931_20), rs1828591 (C__11482211_10), and rs13147758 (C___2965080_10). These SNPs were identified in intronic (non-coding) regions. Supplementary Table S1 summarizes the principal characteristics of the assessed SNPs.
Genotyping was performed by real-time PCR (qPCR) in a StepOne Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific Inc., Singapore) and genotype assignment with sequence detection software (SDS) version 2.3 (Applied Biosystems, CA, United States).

Ortega-Martínez A., Pérez-Rubio G., Ambrocio-Ortiz E., Nava-Quiroz K.J., Hernández-Zenteno R.D., Abarca-Rojano E., Rodríguez-Llamazares S., Hernández-Pérez A., García-Gómez L., Ramírez-Venegas A, & Falfán-Valencia R. (2020). The SNP rs13147758 in the HHIP Gene Is Associated With COPD Susceptibility, Serum, and Sputum Protein Levels in Smokers. Frontiers in Genetics, 11, 882.

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RT-qPCR Assay for Neurodegenerative Diseases

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RT-qPCR assays were done in duplicate on cDNA samples obtained from the retrotranscription reaction and were performed in 384-well optical plates (Applied Biosystems) utilizing the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). 20x TaqMan Gene Expression Assays and 2x TaqMan Universal PCR Master Mix (Applied Biosystems) were used for performing the amplification reactions. TaqMan probes used in this study are shown in Table 2. The selection of these probes was based on parallel studies using the same probes carried out in other neurodegenerative diseases, particularly AD, Parkinson's disease, and Creutzfeldt-Jakob's disease, for future comparative purposes. The reactions were performed following the sequence of temperature and time: 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. TaqMan PCRs were recorded using the Sequence Detection Software (SDS version 2.3, Applied Biosystems). Threshold cycle (CT) data for each sample were analyzed. First, delta CT (ΔCT) values were calculated by normalizing the CT values of each target gene with the endogenous control β-glucuronidase (GUS-β) for normalization [45 (link)]. Second, ΔΔCT values were obtained with the ΔCT of each sample minus the mean ΔCT of the population of control samples (calibrator samples). The fold-change was determined using the equation 2^−ΔΔCT.

Berjaoui S., Povedano M., Garcia-Esparcia P., Carmona M., Aso E, & Ferrer I. (2015). Complex Inflammation mRNA-Related Response in ALS Is Region Dependent. Neural Plasticity, 2015, 573784.

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APOE Genotyping from Whole Blood

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DNA was extracted from whole blood samples; two TaqMan assays (Rs429358 and Rs7412, Assay-On-Demand, Applied Biosystems) were used and run on a 7900HT analyser (Applied Biosystems) and genotypes indicated by the Sequence Detection Software version 2.0 (Applied Biosystems). APOE was modelled as those with and without ε4 allele(s).

Singh-Manoux A., Oumarou-Ibrahim A., Machado-Fragua M.D., Dumurgier J., Brunner E.J., Kivimaki M., Fayosse A, & Sabia S. (2022). Association between kidney function and incidence of dementia: 10-year follow-up of the Whitehall II cohort study. Age and Ageing, 51(1), afab259.

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Quantification of Tissue and Urinary miR-21

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The RNA isolation kit was purchased from Ambion, Inc. The methods of RNA extraction have been described previously.¹⁹ (link) Briefly, ten 10-μm sections were cut from the fresh frozen tissue blocks of the residual sample from the kidney biopsy using a microtome, pooled in a 1.5-mL microcentrifuge tube, and homogenized and dissolved in digestion buffer.²⁰ (link) Urine samples were centrifuged at 3,000g for 30 minutes and 13,000g for 5 minutes at 4 °C. Specimens were then stored at −80 °C until use.
Tissue and urinary miR-21 levels were quantified using real-time quantitative polymerase chain reaction (RT-QPCR) using the ABI Prism 7900 Sequence Detection System (Applied Biosystems). 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). RNU48 (Applied Biosystems) was used as housekeeping genes.²¹ (link)^,²² (link) All RT-QPCR experiments were performed in triplicate. Results were analyzed using Sequence Detection Software, version 2.0 (Applied Biosystems). The ΔΔCT method for relative quantitation was used. Urinary miR-21 level represents the total amount excreted in an 8-hour overnight urine specimen, as compared with that of the housekeeping gene.

Szeto C.C., Ng J.K., Fung W.W., Luk C.C., Wang G., Chow K.M., Lai K.B., Li P.K, & Lai F.M. (2020). Kidney microRNA-21 Expression and Kidney Function in IgA Nephropathy. Kidney Medicine, 3(1), 76-82.e1.

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Quantification of Oncogenic lncRNA Targets

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We performed real-time quantitative polymerase chain reaction (RT-QPCR) using the ABI Prism 7700 Sequence Detector System (Applied Biosystems, Foster City, CA, USA) to determine the lncRNA level. Based on previous studies [11 (link),12 (link),13 (link),14 (link),15 (link),16 (link),17 (link),19 (link),20 (link),21 (link),22 (link),23 (link),24 (link),25 (link),30 (link)], we quantified the lncRNA targets maternally expressed gene 3 (MEG3), antisense non-coding RNA in the INK4 locus (ANRIL), long non-coding mega-cluster (lnc-MGC), metastasis-associated lung carcinoma transcript 1 (MALAT1), cancer susceptibility candidate 2 (CASC2), and taurine-upregulated gene 1 (TUG1). All primer and probe sequences were custom-designed (Applied Biosystems). Small RNA U6 (Applied Biosystems) was used as a house-keeping gene to normalize the lncRNA level [31 (link)]. Results were analyzed with Sequence Detection Software version 2.0 (Applied Biosystems) using the ΔΔCT method for relative quantitation, and results were expressed as copy number per 1000 copies of the housekeeping gene.

Szeto C.C., So H., Poon P.Y., Luk C.C., Ng J.K., Fung W.W., Chan G.C., Chow K.M., Lai F.M, & Tam L.S. (2023). Urinary Long Non-Coding RNA Levels as Biomarkers of Lupus Nephritis. International Journal of Molecular Sciences, 24(14), 11813.

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Influenza A/B Detection and Subtyping

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The one step RT-qPCR (15 (link)) was used to detect universal influenza virus A and B in stools. Influenza A virus specimens underwent H-subtyping using an identical RT-qPCR protocol as employed for influenza A virus testing except for the primers and probes (16 (link)) (Supplementary 2). RT-qPCR testing was performed in a closed tube system using the ABI 7500 fast real-time PCR system (Applied Biosystems) with a 10 μl reaction volume containing 2.5 μl TaqMan Fast Virus 1-Step Master Mix (Thermofisher, ON, Canada), 0.8 μM of each primer, 0.2 μM of probe and 5 μl of NA. PCR reactions included initial incubation at 50°C for 5 minutes and then 95°C for 20 seconds followed by 45× amplification cycles at 95°C for 3 seconds and 60°C for 30 seconds. Amplification data were collected and analyzed with Sequence Detection Software version 2.0 (Applied Biosystems). The C_T cutoff for a positive result was set at 38 for both influenza A/B and H typing.

Xie J., Pang X.L., Tarr G.A., Mu Y., Zhuo R., Chui L., Lee B.E., Vanderkooi O.G., Tarr P.I., Ali S., MacDonald S.E, & Freedman S.B. (2020). Influenza Virus Detection in the Stool of Children with Acute Gastroenteritis. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, 131, 104565.

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Quantification of Urinary RNA Biomarkers

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For the validation cohort, urinary level of RNA targets identified in the previous part were measured by real-time quantitative polymerase chain reaction (RT-QPCR) using the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). We used commercially available unlabeled Taqman® primers and FAM™ dye-labeled TaqMan® MGB probe for the targets (Applied Biosystems), and the RNU48 as the house-keeping gene [10 (link), 11 (link)]. We analyzed the results by Sequence Detection Software version 2.0 (Applied Biosystems). For quantitation, we used the relative ΔΔCT method. As described previously [17 , 18 (link)], when there was no detectable transcript after 40 cycles, we assigned the value half of the detection limit for non-parametric analysis.

Szeto C.C., Wang G., Ng J.K., Kwan B.C., Mac-Moune Lai F., Chow K.M., Luk C.C., Lai K.B, & Li P.K. (2019). Urinary miRNA profile for the diagnosis of IgA nephropathy. BMC Nephrology, 20, 77.

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Standardized APOE Genotyping Protocols

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In the European memory centers, APOE genotype was determined using standard polymerase chain reaction and restriction enzyme digestion according to established standard protocols [21 (link)].
In the ADNI study, APOE genotypes were determined by using DNA extracted by Cogenics from a 3-mL aliquot of EDTA blood. Polymerase chain reaction amplification was followed by HhaI restriction enzyme digestion, resolution on 4% Metaphor Gel, and visualization by ethidium bromide staining [22 (link)].
In Whitehall II and the Three-City Study, two TaqMan assays (Rs429358 and Rs7412, Assay-On-Demand, Applied Biosystems) were used and run on a 7900HT analyzer (Applied Biosystems), and APOE genotypes were indicated by the Sequence Detection Software version 2.0 (Applied Biosystems) [23 (link),24 (link)].

Saddiki H., Fayosse A., Cognat E., Sabia S., Engelborghs S., Wallon D., Alexopoulos P., Blennow K., Zetterberg H., Parnetti L., Zerr I., Hermann P., Gabelle A., Boada M., Orellana A., de Rojas I., Lilamand M., Bjerke M., Van Broeckhoven C., Farotti L., Salvadori N., Diehl-Schmid J., Grimmer T., Hourregue C., Dugravot A., Nicolas G., Laplanche J.L., Lehmann S., Bouaziz-Amar E., Hugon J., Tzourio C., Singh-Manoux A., Paquet C, & Dumurgier J. (2020). Age and the association between apolipoprotein E genotype and Alzheimer disease: A cerebrospinal fluid biomarker–based case–control study. PLoS Medicine, 17(8), e1003289.

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