Ssoadvanced universal supermix

Manufactured by Bio-Rad

Sourced in Japan, United States

SsoAdvanced Universal Supermix is a ready-to-use solution for quantitative PCR (qPCR) reactions. It contains all the necessary components, including DNA polymerase, dNTPs, and buffers, to perform efficient and sensitive qPCR amplification.

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Lab products found in correlation

Rneasy mini kit, by Qiagen (3 mentions) Cfx96 real time system, by Bio-Rad (3 mentions) High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions) Cfx manager software, by Bio-Rad (2 mentions) Cfx96 real time pcr detection system, by Bio-Rad (1 mentions) Maxima universal first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Cfx96, by Bio-Rad (1 mentions) Dneasy plant mini kit, by Qiagen (1 mentions) Direct zol rna kit, by Zymo Research (1 mentions) Qscript cdna supermix, by Quanta Biosciences (1 mentions) Primepcr probes, by Bio-Rad (1 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions) Primepcr assay, by Bio-Rad (1 mentions) Miniopticon real time pcr system, by Bio-Rad (1 mentions) Quantitect reverse transcription kit, by Qiagen (1 mentions)

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11 protocols using ssoadvanced universal supermix

Quantitative Gene Expression Analysis

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First-strand cDNA was prepared from 1 μg of total RNA with the Maxima Universal First Strand cDNA Synthesis kit (Thermo Fisher Scientific, www.thermofisher.com/) and diluted 1:4 in RNase-free water prior to use. Transcript levels were determined with qPCR using Bio-Rad SsoAdvanced Universal Supermix and the CFX96 Real-Time PCR Detection System according to manufacturer’s protocols (Bio-Rad, www.bio-rad.com). Transcript levels of target genes were assessed using normalized relative quantity (NRQ) calculated from the Pfaffl method using the geometric mean of two reference gene quantities [80 (link), 81 (link)]. Reference genes were PP2A (At1g13320) and IPP2 (At3g02780) [82 (link)]. Primer sequences for all reference and target genes are in Additional file 8: Table S3.

Gonzalez L.E., Keller K., Chan K.X., Gessel M.M, & Thines B.C. (2017). Transcriptome analysis uncovers Arabidopsis F-BOX STRESS INDUCED 1 as a regulator of jasmonic acid and abscisic acid stress gene expression. BMC Genomics, 18, 533.

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Quantifying Messenger RNA Expression

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RNA was first isolated using the RNeasy Mini Kit (QIAGEN) and then reverse transcribed using the High-Capacity RNA-to-cDNA kit (Thermo Fisher Scientific) according to the manufacturers’ instructions. RT-qPCR was performed using the SsoAdvanced Universal Supermix (Biorad) with Quantitect primers (TPP1 only) or previously validated primers^{20 (link),27 (link)}. Primer sequences available upon request.

Ruis P., Van Ly D., Borel V., Kafer G.R., McCarthy A., Howell S., Blassberg R., Snijders A.P., Briscoe J., Niakan K.K., Marzec P., Cesare A.J, & Boulton S.J. (2020). TRF2-independent chromosome end protection during pluripotency. Nature, 589(7840), 103-109.

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RT-qPCR quantification of BLM and GAPDH

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RNA was first isolated using the RNeasy Mini Kit (QIAGEN) and then reverse transcribed using the High-Capacity RNA-to-cDNA kit (Thermo Fisher Scientific) according to the manufacturers’ instructions. RT-qPCR was performed with the following primers: BLM TaqMan probe_Hs00172060_m1 (Cat# 4331182, ThermoFisher) and GAPDH TaqMan probe_Hs02758991_g1 (Cat# 4448484, ThermoFisher) using the SsoAdvanced Universal Supermix (Biorad).

Panier S., Maric M., Hewitt G., Mason-Osann E., Gali H., Dai A., Labadorf A., Guervilly J.H., Ruis P., Segura-Bayona S., Belan O., Marzec P., Gaillard P.H., Flynn R.L, & Boulton S.J. (2019). SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance. Molecular Cell, 76(1), 27-43.e11.

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Quantifying Puccinia graminis in Wheat

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DNA was extracted from 100 mg of fresh roots using DNeasy Plant Mini Kit (Qiagen, Tokyo, Japan) using the suppliers’ protocols. The TaqMan assay used to quantify P. graminis was targeted to the rDNA ITS using primers and probes described elsewhere (Liu et al., 2016 (link)) and listed in Table 1. Each 10-µl reaction contained 2× Sso Advanced Universal Supermix (Bio-Rad, Tokyo, Japan), 10 ng DNA template, 300 nM of either P. graminis rDNA ITS or wheat actin primers, and 300 nM of either P. graminis rDNA ITS or wheat actin probe. The reaction was initiated by a denaturing (95°C/30 s) step, which was followed by 50 cycles of 95°C/10 s and 55°C/30 s using a CFX96 real-time system device (Bio-Rad). The baseline threshold was set to 100 for both FAM and Texas Red. Two technical replicates were used for each plant, along with three biological replicates per treatment. The Tukey–Kramer method was applied to find multiple-sample means that are significantly different from each other.

Okada K., Xu W., Mishina K., Oono Y., Kato T., Namai K, & Komatsuda T. (2023). Genetic resistance in barley against Japanese soil-borne wheat mosaic virus functions in the roots. Frontiers in Plant Science, 14, 1149752.

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Quantitative Analysis of Telomere and DNA Damage Response Transcripts

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Total RNA was extracted from plant tissues using a Directzol RNA kit (Zymo Research). Reverse transcription was performed with 1 μg total RNA with the qScript cDNA SuperMix (Quanta Biosciences). mRNA levels were assessed by quantitative PCR with primers described previously to detect telomere-related transcripts (Cifuentes-Rojas et al. 2011 (link); Leehy et al. 2013 (link)) or DDR transcripts (Boltz et al. 2012 (link); Cifuentes-Rojas et al. 2012 (link); Hashimura and Ueguchi 2011 (link)), using SsoAdvanced Universal Supermix (Bio-Rad). RNA from at least three individual plants was used for each genetic background and at least two technical replicates were run for each reaction. Expression levels were averaged and normalized to GAPDH. Wild-type was set to 1 and mutant samples were compared to this value. To detect transcription of retrotransposons, primers recognizing AtMu1 and ATGP3 were used as described previously (Hirochika et al. 2000 (link); Tsukahara et al. 2009 (link)).
TERRA was monitored by northern blot using 10 μg of total RNA. RNA was resolved on a 1% agarose gel, transferred to a nylon membrane and hybridized with a ³²P 5′ labeled (CCC TAA A)₅ probe as previously described (Zellinger et al. 2007 (link)).

Xie X, & Shippen D.E. (2018). DDM1 guards against telomere truncation in Arabidopsis. Plant cell reports, 37(3), 501-513.

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Quantitative PCR Assay Optimization

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Quantitative PCR was performed with SsoAdvanced Universal Supermix (Bio-Rad, Hercules, CA, USA) on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). A melting curve was performed after each PCR reaction to confirm the specificity of the primers. All reactions were performed in duplicate. Data were analyzed using CFX Manager Software (Bio-Rad, Hercules, CA, USA).

Vitiello M., Mercatanti A., Podda M.S., Baldanzi C., Prantera A., Sarti S., Rizzo M., Salvetti A., Conte F., Fiscon G., Paci P, & Poliseno L. (2023). A Network of MicroRNAs and mRNAs Involved in Melanosome Maturation and Trafficking Defines the Lower Response of Pigmentable Melanoma Cells to Targeted Therapy. Cancers, 15(3), 894.

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RNA Extraction and qPCR Analysis

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RNA was extracted using an RNeasy Mini Kit (Qiagen). qPCR was performed using a PrimePCR Probe Assay consisting of iScript cDNA synthesis, PrimePCR Probes (PrimePCR™ Probe Assay: Slc23a1, Mouse), and Sso Advanced Universal Supermix (Bio Rad).

Consoli D.C., Jesse J.J., Klimo K.R., Tienda A.A., Putz N.D., Bastarache J.A, & Harrison F.E. (2020). A Cecal Slurry Mouse Model of Sepsis Leads to Acute Consumption of Vitamin C in the Brain. Nutrients, 12(4), 911.

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Glomerular RNA Extraction and qPCR Analysis

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Glomeruli were captured from OCT-embedded, cryosectioned kidney samples using PALM laser-capturing microdissection system (Carl Zeiss microImaging GmbH, Munich, Germany) as previously described [34 (link), 35 ]. Total RNA was extracted with RNeasy kit (Quagen, Valencia, CA) as instructed by the manufacturer, and then reverse-transcribed with the iScript cDNA synthesis kit (Bio-Rad Laboratories) for quantitative PCR using the SsoAdvanced Universal Supermix (Bio-Rad Laboratories) with the CFX96 Touch real-time PCR detection system (Bio-Rad). Real-time data were collected for 40 cycles of 95°C, 10 s, 57°C, 45 s, and 75°C, 30 s. Samples were run in duplicates and relative expression of the gene of interest was estimated by the ∆∆Ct method using 18S as a reference gene. Primers used were custom-synthesized by Integrated DNA Technology (Coralville, IA) and detailed primer information is provided in Table 1.

Liang X., Schnaper H.W., Matsusaka T., Pastan I., Ledbetter S, & Hayashida T. (2016). Anti-TGF-β Antibody, 1D11, Ameliorates Glomerular Fibrosis in Mouse Models after the Onset of Proteinuria. PLoS ONE, 11(5), e0155534.

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Quantitative RT-PCR Analysis of Gene Expression

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1 μL of cDNA and appropriate primers (Table 1) were used for qRT-PCR reactions, using SsoAdvanced Universal Supermix (Bio-Rad; Hercules, California) on a CFX96 Real-Time System (Bio-Rad; Hercules, California). The reaction conditions were the following: 98 °C 30 sec, (98 °C 3 sec, 60,4 °C 20 sec, 72 °C 10 sec)x39cycles. In order to confirm the specificity of the reaction, a melting curve was performed after each PCR (from 65 °C to 95 °C with an increase of temperature of 0.5 °C/sec). The annealing temperature was optimized by performing a gradient with each primer pair (from from 55 °C to 64 °C). All reactions were performed in duplicate; the raw values were calculated using three housekeeping genes as reference (GAPDH, SDHA, PBGD) according to the ΔΔCq method used by the CFX Manager Software (BioRad).

Capobianco E., Valdes C., Sarti S., Jiang Z., Poliseno L, & Tsinoremas N.F. (2017). Ensemble Modeling Approach Targeting Heterogeneous RNA-Seq data: Application to Melanoma Pseudogenes. Scientific Reports, 7, 17344.

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Validating miR-203a-3p Target Genes

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miRWalk 2.0 was used to predict mRNAs targeted by miR-203a-3p (32 (link)). Four target genes known to be associated with metastasis in solid tumors [Actin, gamma 2, smooth muscle, enteric (ACTG2), calponin 1 (CNN1), major histocompatibility complex, class II, DP beta 1 (HLA-DPB1) and myosin light chain kinase (MYLK)] were selected for analysis by RT-qPCR (33 (link)). The expression of these genes was quantified in the same cellular MDA-MB-436 samples previously used for NGS. Initially, RNA was reverse transcribed in triplicate using the QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer's instructions. Quantitative PCR was then performed using Prime PCR Assays, SsoAdvanced Universal Supermix (Bio-Rad) and 10 ng of template cDNA. PCR reactions were run on a MiniOpticon real-time PCR system (Bio-Rad). Target gene Cq values were normalized to GAPDH (19 (link)).

Buschmann D., González R., Kirchner B., Mazzone C., Pfaffl M.W., Schelling G., Steinlein O, & Reithmair M. (2018). Glucocorticoid receptor overexpression slightly shifts microRNA expression patterns in triple-negative breast cancer. International Journal of Oncology, 52(6), 1765-1776.

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