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Absolute blue qpcr mix

Manufactured by Thermo Fisher Scientific
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Absolute Blue qPCR Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) applications. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, and buffer, optimized for efficient and reliable qPCR reactions.

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Lab products found in correlation

Epoch spectrophotometer, by Agilent Technologies (5 mentions) 7000 real time pcr system, by Thermo Fisher Scientific (4 mentions) Dneasy column, by Qiagen (4 mentions) Iscript cdna synthesis kit, by Bio-Rad (4 mentions) 2100 bioanalyzer, by Agilent Technologies (4 mentions) Rneasy fibrous tissue mini kit, by Qiagen (4 mentions) Dnase 1, by Thermo Fisher Scientific (4 mentions) Verso cdna synthesis kit, by Thermo Fisher Scientific (3 mentions) Stratagene mx3005p, by Agilent Technologies (3 mentions) T100 thermal cycler, by Bio-Rad (3 mentions) Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions) Viia7, by Thermo Fisher Scientific (3 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions) Turbo dna free kit, by Thermo Fisher Scientific (3 mentions) Superscript 3, by Thermo Fisher Scientific (2 mentions) Universal probe library, by Roche (2 mentions) 7300 real time pcr instrument, by Thermo Fisher Scientific (2 mentions) Faststart universal probe library master mix, by Roche (2 mentions) Taqman microrna assay kit, by Thermo Fisher Scientific (2 mentions) High capacity cdna rt kit, by Thermo Fisher Scientific (2 mentions)

36 protocols using absolute blue qpcr mix

1

qPCR Assay for MMP-2 and MMP-3 Expression

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The qPCR conditions used have been previously described [10 (link)]. Briefly, total RNA (200 ng) from each sample was reverse transcribed using Superscript III (Invitrogen). MMP-2 (forward-CGGTTTTCTCGAATCCATGA; reverse-GGTATCCATCGCCATGCT) and MMP-3 (forward-CCAGGTGTGGAGTTCCTGA; reverse-GCATCTTTTGGCAAATCTGG) primers were designed based on the Universal ProbeLibrary (Roche, Indianapolis, IN). Reactions were prepared with Absolute Blue qPCR Mix (Thermo Fisher, Waltham, MA), and run in duplicate on a LightCycler 480 thermocycler (Roche) using Relative Quantitative Software (Roche). Ct (threshold cycle) values were adjusted for GAPDH in each sample (ΔCt). Fold differences were calculated with the 2−ΔΔCt method [22 (link)].
Laragione T., Cheng K.F., Tanner M.R., He M., Beeton C., Al-Abed Y, & Gulko P.S. (2015). THE CATION CHANNEL TRPV2 IS A NEW SUPPRESSOR OF ARTHRITIS SEVERITY, JOINT DAMAGE AND SYNOVIAL FIBROBLAST INVASION. Clinical immunology (Orlando, Fla.), 158(2), 183-192.
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2

Quantitative Real-Time PCR for rAAV Vector Genomes

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Total DNA was isolated from tissue samples using QIAGEN DNeasy columns. The DNA samples were assayed in duplicates by quantitative real-time PCR, using ABsolute Blue qPCR mix (Thermo Scientific) and an Applied Biosystems 7000 real-time PCR system, following the procedures recommended by the manufacturers. TaqMan primers specific for hSGSH were used to detect rAAV vector genomes as follows: forward, 5′-AAGTCAGCGAGGCCTACGT-3′; reverse, 5′-GATGGTCTTCGAGCCAAAGAT-3′; probe, 5′-(6-FAM)-CCTCCTAGACCTCACGCCCAC C-(TAMRA)-3′. Genomic DNA (gDNA) was quantified in parallel samples using mouse β-actin-specific primers as follows: forward, 5′-GTCATCACTAT TGGCAACGA-3′; reverse. 5′-CTCAGGA GTTTTGTCACCTT-3′, probe: 5′-(6-FAM)-TTCCGATG CCCTGAGGCTCT-(TAMRA)-3′. Serial diluted vector plasmid and mouse gDNA were used as standards. gDNA from tissues of non-treated mice were used as controls for background levels and absence of contamination. Data are expressed as vg/μg gDNA.
Bobo T.A., Samowitz P.N., Robinson M.I, & Fu H. (2020). Targeting the Root Cause of Mucopolysaccharidosis IIIA with a New scAAV9 Gene Replacement Vector. Molecular Therapy. Methods & Clinical Development, 19, 474-485.
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3

miRNA Quantification via RT-qPCR

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For miRNA-specific complementary DNA synthesis, 10 ng total RNA was reversely transcribed using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster city, CA, USA) in combination with multiplexed reverse transcription primers of miR-625-3p (TaqMan, Applied Biosystems). Single assay real-time-PCR for specific miRNAs was performed using 10 ng total cDNA with ABsolute Blue qPCR Mix (Thermo Scientific) and specific miRNA TaqMan Probes. RNU48 and U6 snRNA were used as internal controls for normalization using the ratio relative quantity (RQ) target gene/RQ normalization gene. RQ values were generated by the ViiA7 (Applied Biosystems) Real Time PCR instrument, using the formula RQ = 10[(ct-b)/a] (where ct is the threshold cycle, a and b are slope and intersection respectively, generated from a relative standard curve plotted from a pool of randomly selected samples).
Verma K., Jyotsana N., Buenting I., Luther S., Pfanne A., Thum T., Ganser A., Heuser M., Weissinger E.M, & Hambach L. (2017). miR-625-3p is upregulated in CD8+ T cells during early immune reconstitution after allogeneic stem cell transplantation. PLoS ONE, 12(8), e0183828.
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4

Quantitative RT-PCR Analysis of ATF5

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Total RNA was extracted using Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada) and reversed transcribed into cDNA using Verso cDNA Synthesis Kit (ThermoFisher Scientific). Real-time PCR was performed on a Rotor-Gene 6000 (Qiagen, Hilden, Germany) or Eco Real-time PCR System (Illumina, San Diego, CA, USA), using Absolute Blue QPCR Mix (ThermoFisher Scientific) according to the manufacturer’s recommendations. Amplification specificity was verified by melting curve analysis. Values of mRNA expression were normalized to the level of B2M expression. The oligonucleotide primers used were as follows:

ATF5 sense 5′-AATTGAGGTGTATAAGGCCCG-3′

ATF5 antisense 5′-GGATAGGAAAGTGGAATGGAGG-3′

B2M sense 5′-TTCTGGTGCTTGTCTCACTGA-3′

B2M antisense 5′-CAGTATGTTCGGCTTCCCATTC-3′

Ben-Shmuel S., Rashed R., Rostoker R., Isakov E., Shen-Orr Z, & LeRoith D. (2017). Activating Transcription Factor-5 Knockdown Reduces Aggressiveness of Mammary Tumor Cells and Attenuates Mammary Tumor Growth. Frontiers in Endocrinology, 8, 173.
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5

Quantitative PCR Analysis of Cln3 and Gapdh

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Using nuclease-free water, cDNA samples were diluted 1:20. Quantitative polymerase chain reactions were performed using Absolute Blue Q-PCR Mix (Thermo Fisher Scientific). Cln3 (Forward primer: 5′-TGGAGACCAGTGACAAGCA-3′; Reverse primer: 5′-TCAAGGGAGGTGACAGAGGA-3′) and Gapdh expression (Forward primer: 5′-ACCACAGTCCATGCCATCAC-3′; Reverse primer: 5′-TCCACCACCCTGTTGCTGTA-3′) were quantified. Reactions were performed in a Stratagene Mx3005P (Agilent Technologies) under the following conditions: 1) 95 °C for 15 min. (1 cycle) and 2) 95 °C for 15 s.; 60 °C for 1 min. (40 cycles). 2-ΔΔCT method was used to analyze relative fold expression65 (link).
Langin L., Johnson T.B., Kovács A.D., Pearce D.A, & Weimer J.M. (2020). A tailored Cln3Q352X mouse model for testing therapeutic interventions in CLN3 Batten disease. Scientific Reports, 10, 10591.
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6

RNA Extraction and qPCR for Gene Expression Analysis

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Total RNA was extracted using ZR RNA MiniPrep kit (Zymo Research) or TRIzol (Sigma-Aldrich) with DNase I (Thermo Scientific). cDNA was prepared using High Capacity cDNA RT Kit (Applied Biosystems). qPCR was carried out using ABsolute blue qPCR Mix (Thermo Scientific) or FastStart Universal Probe Library Master Mix (Roche) in a 7300 real-time PCR instrument (Applied Biosystems). The results were normalized to transcripts of TATA-box-binding protein (TBP) and human large ribosomal protein (RPLPO). Table 2 lists primer sequences designed for Universal Probe Library (Roche). All reactions were performed with annealing at 60°C for 40 cycles. For undetectable transcripts, the cycle number was set to 40 for comparisons. cDNA for miRNA analyses was prepared and analyzed using Taqman MicroRNA Assay kit (Applied Biosystems) according to the manufacturer, with primers listed in Table 3.
Nathan G., Kredo-Russo S., Geiger T., Lenz A., Kaspi H., Hornstein E, & Efrat S. (2015). MiR-375 Promotes Redifferentiation of Adult Human β Cells Expanded In Vitro. PLoS ONE, 10(4), e0122108.
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7

RNA Extraction and qRT-PCR Analysis

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Samples harvested in TRIzol were homogenized using a 21-gauge needle. Chloroform was added to the TRIzol samples (1 in 5 v/v) and separation was achieved following centrifugation at 12,000 g for 15 minutes. The upper aqueous RNA phase was purified using DNase I and an RNeasy Mini Kit (Qiagen NV, Venlo, the Netherlands) according to the manufacturers’ instructions.
cDNA was synthesized from 50 ng RNA using the Verso™ 2-step qRT-PCR kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed using the Stratagene machine (Agilent Technologies, Santa Clara, CA, USA). cDNA (1 μL) was added to ABsolute Blue qPCR mix (Thermo Fisher Scientific) according to the manufacturers’ instructions in the presence of 6-carboxyfluorescein (FAM)-labeled primers for CXCL8, TNFα, TLR1, TLR2, TLR4, or GAPDH (Thermo Fisher Scientific). Amplification conditions were 95°C for 15 minutes, 95°C for 15 minutes before cooling to 60°C for 1 minute for 40 cycles. Levels of CXCL8, TNFα, TLR1, TLR2, and TLR4 were normalized to housekeeping gene GAPDH to quantify mRNA levels.
Lea S.R., Reynolds S.L., Kaur M., Simpson K.D., Hall S.R., Hessel E.M, & Singh D. (2018). The effects of repeated Toll-like receptors 2 and 4 stimulation in COPD alveolar macrophages. International Journal of Chronic Obstructive Pulmonary Disease, 13, 771-780.
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8

Quantitative RT-qPCR for Meiotic and Mitotic Gene Expression

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Five μg of isolated total RNA was treated with DNase (TURBO DNA-free Kit). cDNA was reverse transcribed following the Superscript III kit (Thermo Fisher Scientific). Quantification was performed with Absolute Blue qPCR Mix (Thermo Fisher Scientific). Meiotic samples were normalized to PFY1, and mitotic samples were normalized to ACT1. Oligonucleotides are listed in Supplementary file 6.
Ünal E., Su A.J, & Yendluri S.C. (2024). Control of meiotic entry by dual inhibition of a key mitotic transcription factor. eLife, 12, RP90425.
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9

Quantitative Real-Time PCR for Gene Expression

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Prior to their use, cDNA samples were diluted 1:20 using nuclease free water. Quantitative real-time polymerase chain reactions were carried out using the Absolute Blue QPCR Mix (Thermo Fisher Scientific). Cln2 expression was measured using the Tpp1 TaqMan Gene Expression Assay from Thermo Fisher Scientific (Mm00487016_m1). In addition, Gapdh expression was measured using the Gapdh TaqMan Gene Expression Assay from Thermo Fisher Scientific (Mm99999915_g1). All reactions were run in a Stratagene Mx3005P qPCR machine (Agilent Technologies). Reaction conditions consisted of the following: 1) 95°C for 15 min. (1 cycle) and 2) 95°C for 15 sec.; 60°C for 1 min. (40 cycles). ΔΔCT was used to calculate relative fold expression.
Geraets R.D., Langin L.M., Cain J.T., Parker C.M., Beraldi R., Kovacs A.D., Weimer J.M, & Pearce D.A. (2017). A tailored mouse model of CLN2 disease: A nonsense mutant for testing personalized therapies. PLoS ONE, 12(5), e0176526.
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10

Quantitative RT-PCR Analysis of Blood RNA

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Total blood RNA from each study subject was assayed by qRT-PCR for gene expression using sequence-specific primers and probes, to confirm the quality and accuracy of microarray data. Reverse transcriptase reactions were performed using the Superscript III First Strand Synthesis Kit (Life Technologies, CA). Quantitative real-time PCR reactions were performed using Absolute Blue QPCR Mix (Thermo Scientific, MA) and Applied Biosystems 7000 Real-Time PCR System (Life technologies, CA) following the procedures recommended by the manufacturers. Primers and probes used are listed in Supplemental Table S1. All reactions were run in triplicate as a duplex reaction, with probes conjugated to either Fam or Hex (Sigma, MO). Primers for human GAPDH were used as an internal control because no change was observed in GAPDH transcripts in AD blood using arrays. The levels of target mRNAs relative to GAPDH mRNA were determined, as based on the on-plate standard curve, and presented as patients vs. controls.
Naughton B.J., Duncan F.J., Murrey D.A., Meadows A.S., Newsom D.E., Stoicea N., White P., Scharre D.W., Mccarty D.M, & Fu H. (2015). Blood genome-wide transcriptional profiles reflect broad molecular impairments and strong blood-brain links in Alzheimer’s disease. Journal of Alzheimer's disease : JAD, 43(1), 93-108.
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