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Sso advanced universal probes mastermix

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The Sso Advanced Universal Probes Mastermix is a reagent formulation designed for real-time PCR applications. It contains a proprietary blend of DNA polymerase, buffer, and dNTPs optimized for use with hydrolysis probe-based detection.

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Lab products found in correlation

High capacity reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Peqgold total rna kit, by Avantor (1 mentions) Iscript gdna clear cdna synthesis kit, by Bio-Rad (1 mentions) Rnaeasy midi kit, by Qiagen (1 mentions) Taqman primer probe mix, by Thermo Fisher Scientific (1 mentions) Dnase, by Thermo Fisher Scientific (1 mentions) Taqman preamp kit, by Thermo Fisher Scientific (1 mentions)

4 protocols using sso advanced universal probes mastermix

1

Quantitative Analysis of Gene Expression and Viral RNA

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Gene expression and viral RNA were quantified by RT-qPCR. Total RNA was isolated using a peqGOLD Total RNA Kit (VWR, Radnor) and 100 ng RNA was reverse transcribed using iScript gDNA Clear cDNA Synthesis Kit (BioRad, Hercules). For qPCR analysis, cDNA was diluted 1:5. qPCR was then performed using Sso Advanced Universal Probes Mastermix (BioRad, Hercules) on a StepOne Plus Real-Time PCR system (ThermoFisher, Waltham). Oligonucleotide sequences are listed in the Supplementary Information (Table S2). For gene expression analysis, fold changes relative to uninfected controls were calculated using the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)) with GAPDH as normalization gene. Intracellular viral RNA copies were quantified using a standard curve derived from synthetic double-stranded DNA.
Endres A., Hügel C., Boland H., Hogardt M., Schubert R., Jonigk D., Braubach P., Rohde G, & Bellinghausen C. (2022). Pseudomonas aeruginosa Affects Airway Epithelial Response and Barrier Function During Rhinovirus Infection. Frontiers in Cellular and Infection Microbiology, 12, 846828.
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2

Allele-specific PCR for BRAF V600E Detection

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Allele-specific PCR was a modified version of the allele-specific locked nucleic acid PCR published by Morandi et al.41 (link). Briefly, each qPCR reaction included 10 µl of DNA eluate or 10 ng of genomic DNA as positive control (Cat Num. HD238, Horizon Discovery), 1X SsoAdvanced Universal Probes Mastermix (Cat Num. 172-5285, Bio-Rad, US), 0.625 µl of primers (10 µM) and 0.3125 µl of fluorescent probe (10 µM) in a total volume of 25 µl. After careful mixing, each reaction was loaded in triplicate on a 96-well PCR plate and the following qPCR program was launched: 95 °C for 3′, 40 cycles at 95 °C for 5″ and 60 °C for 30″. To calculate the % of recovered BRAFV600E gene the following formula was used: = [2^(Ct input – Ct isolation)]%
Zocco D., Bernardi S., Novelli M., Astrua C., Fava P., Zarovni N., Carpi F.M., Bianciardi L., Malavenda O., Quaglino P., Foroni C., Russo D., Chiesi A, & Fierro M.T. (2020). Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients. Scientific Reports, 10, 15745.
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3

Oprk1 Expression Analysis in Mice

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Brains were extracted from male and female WT and heterozygous Oprk1-Cre mice (age range, ∼8–12 weeks) and flash frozen. Brains were homogenized using a Dounce homogenizer, and total RNA was isolated using the RNAEasy Midi Kit (Qiagen) following manufacturer instructions. RNA was treated with DNase (Ambion). Total RNA was quantified on a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). RNA (1 mg) was reverse transcribed using the High-Capacity Reverse Transcription Kit from Applied Biosystems. cDNA was diluted 1:5, and 2 µl of that solution were subjected to real-time PCR amplification to detect Oprk1 (Mm01230885_m1, Thermo Fisher Scientific) with 5 µl of 1× SSoAdvanced Universal Probes Master Mix (BIO-RAD), 0.5 μl of 20× TaqMan Primer/Probe Mix (Applied Biosystems) in a total volume of 10 μl. Data were normalized to the endogenous control genes for transferrin (Tfrc; Mm00441941_m1, Thermo Fisher Scientific) or gusducin B (GusB; Mm019768_m1, Thermo Fisher Scientific).
Paliarin F., Duplantis C., Jones A.F., Cucinello-Ragland J., Basavanhalli S., Blaze E., Doré E., Neel A.I., Sun H., Chen R., Edwards S., Gilpin N.W., Messing R.O, & Maiya R. (2023). A Cre Driver Line for Genetic Targeting of Kappa Opioid Receptor Expressing Cells. eNeuro, 10(7), ENEURO.0043-23.2023.
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4

Gene Expression Analysis in Lmo4gt/+ Mice

Cited 2 times
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RNA was extracted from wild type and Lmo4gt/+ mice as outlined above. The extracted RNA was amplified using the Taqman PreAmp kit from Applied Biosystems (Thermo Fisher Scientific, Rockford, IL) according to manufacturer’s instruction. Sixty nanograms of RNA were reverse transcribed using the High Capacity Reverse Transcription kit from Applied Biosystems (Thermo fisher Scientific). The preamplification reaction comprised of 15ng of reverse transcribed cDNA, 0.2X Taqman probe, and 1X Taqman PreAmp Mastermix. The preamplified product was diluted 1:20 in 1X TE buffer and 2.5μl of the preamplified product was used in a real time PCR amplification reaction which also contained 5μl of 1X SSoAdvanced Universal Probes Mastermix (Biorad, Hercules, CA), 0.5μl of 20X Taqman primer/probe mix in a total volume of 10μl. Relative mRNA levels of target genes were determined using BIORAD software as previously described (19 (link), 20 (link)). Data were normalized to multiple endogenous control genes including GusB, Tfrc, Tubb2b, and Rplp0. Catalog numbers of Taqman gene expression assays used are listed in Table S3.
Maiya R., Pomrenze M.B., Tran T., Tiwari G.R., Beckham A., Paul M.T., Mayfield R.D, & Messing R.O. (2020). Differential Regulation of Alcohol Consumption and Reward by the Transcriptional Cofactor LMO4. Molecular psychiatry, 26(6), 2175-2186.
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