Perfecta preamp supermix

Manufactured by Quantabio

Sourced in United States

PerfeCTa PreAmp Supermix is a pre-amplification reagent designed for use in quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) applications. The product provides consistent, reliable amplification of target sequences from low-copy-number samples.

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9 protocols using perfecta preamp supermix

Endothelial Cell Isolation and Gene Expression

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Brachial endothelial cell pellets were re-suspended in isolation buffer, incubated with biotinylated mouse anti-human monoclonal antibody directed against CD146 (1:200; Millipore Sigma) and isolated with streptavidin magnetic FlowComp Dynabeads (1:100). Endothelial cells underwent mRNA extraction using RNAqueous – micro RNA isolation kit (Invitrogen, Carlsbad CA). Messenger RNA was converted to cDNA (Quantbio, Beverly, MA) and amplified via PerfeCta PreAmp SuperMix (Quantabio). TaqMan (Life Technologies) primers were used on an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA). To ensure reproducibility across analysis, results are represented as normalized to human acidic ribosomal protein (hARP) for each sample and gene.^{26 (link)}

Garshick M.S., Barrett T., Wechter T., Azarchi S., Scher J., Neimann A., Katz S., Fuentes-Duculan J., Cannizzaro M.V., Jelic S., Fisher E.A., Krueger J.G, & Berger J.S. (2019). Inflammasome Signaling and Impaired Vascular Health in Psoriasis. Arteriosclerosis, thrombosis, and vascular biology, 39(4), 787-798.

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Single-cell RNA Amplification and Quantification

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Following RNA extraction, individual neuron RNA samples were reverse transcribed into cDNA using qScript cDNA SuperMix (QuantaBio, Beverly, MA) primed with random hexamers and oligo-dT per the manufacturer’s protocol in 20-µL reactions. Half of each resulting cDNA pool (10 µL) was preamplified using PerfeCTa PreAmp Supermix (QuantaBio) with a 14-cycle RT-PCR primed with a pool of target-specific primers (SI Appendix, Table S7) in a 20-µL reaction per the manufacturer’s protocol to allow for enough product to carry out 15 multiplex qPCR reactions per individual neuron sample. Amplified and unamplified target abundances were compared to ensure minimal amplification bias in the preamplification of samples (SI Appendix, Fig. S2).

Northcutt A.J., Kick D.R., Otopalik A.G., Goetz B.M., Harris R.M., Santin J.M., Hofmann H.A., Marder E, & Schulz D.J. (2019). Molecular profiling of single neurons of known identity in two ganglia from the crab Cancer borealis. Proceedings of the National Academy of Sciences of the United States of America, 116(52), 26980-26990.

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RNA Extraction and Reverse Transcription

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Total RNA was isolated from each neuron using the Quick-RNA MicroPrep kit (Zymo Research, Irvine, CA, USA) per the manufacturer’s instructions. Reverse transcription of total RNA was then performed using a mixture of oligo-dT and random hexamer primers (qScript cDNA Supermix; QuantaBio, Beverly, MA, USA). Half of the cDNA produced from each neuron (10 μL) was then pre-amplified using PerfeCTa PreAmp Supermix (QuantaBio, Beverly, MA, USA) according to the manufacturer’s instructions (20 μL reaction volume). This protocol utilizes a 14-cycle PCR reaction primed with a pool of target-specific primers to enrich subsequent qPCR reactions when starting sample is limited. After preamplification, cDNA samples were diluted 7.5x in nuclease-free water (150 μL final volume).

Santin J.M, & Schulz D.J. (2019). Membrane voltage is a direct feedback signal that influences correlated ion channel expression in neurons. Current biology : CB, 29(10), 1683-1688.e2.

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Comprehensive RNA Quantification Protocol

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Total RNA was isolated using TRIzol reagent (Thermo Fisher) following the manufacturer's recommendations. cDNA was synthesized using qScript cDNA supermix (Quantabio). Real-time PCR was performed using SYBR Green PCR master mix (Life Technologies) with primers listed in Supplemental Table S1. L32 was used to normalize expression. TaqMan assays (Life Technologies) for miRNA qPCR were normalized to miR-16. The 7500 Fast real-time PCR system (Applied Biosystems) were used for qPCR reactions. Low-abundance cytokines and chemokines were amplified via qPCR by using the PerfeCTa PreAmp supermix (Quantabio). cDNA was synthesized using qScript XLT cDNA supermix (Quantabio). Primers were pooled, and preamplification reactions with PerfeCTa PreAmp supermix were performed following the manufacturer's recommendations. Real-time PCR was performed using iTaq universal SYBR Green supermix (Bio-Rad) with primers listed in Supplemental Table S1. L32 was used to normalize expression. The CFX384 real-time PCR detection system (Bio-Rad) was used.

Kundu S.T., Rodriguez B.L., Gibson L.A., Warner A.N., Perez M.G., Bajaj R., Fradette J.J., Class C.A., Solis L.M., Rojas Alvarez F.R., Wistuba I.I., Diao L., Chen F., Sachdeva M., Wang J., Kirsch D.G., Creighton C.J, & Gibbons D.L. (2022). The microRNA-183/96/182 cluster inhibits lung cancer progression and metastasis by inducing an interleukin-2-mediated antitumor CD8+ cytotoxic T-cell response. Genes & Development, 36(9-10), 582-600.

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RNA and DNA Isolation Followed by RT-qPCR Analysis

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Once excised, each gel pallet was transferred to one centrifuge tube, immediately followed by the addition of 20 μL of DNA/RNA Shield^™ (R1100, Zymo). Sample were stored in −80°C until RNA preparation. RNA and DNA were isolated following the manufacturer’s protocol. Nucleic acids were eluted in 8 μL of water. Alternate mRNA and DNA isolation can be performed with Direct-zol RNA Miniprep Plus (Cat. R2070S, LOT: ZRC202000), RNA Clean & Concentrator^™−5 Cat R1015S (10 preps), LOT: ZRC200969). All 8 μL of RNA sample was used for cDNA synthesis. Reverse transcription of mRNA to cDNA was accomplished with SuperScript IV First Strand Synthesis System (18091050, Thermo Fisher) as per manufacturer instructions. Pre-amplification was done on the resulting 20-μl cDNA sample using the Perfecta PreAmp SuperMix (95146 QuantaBio) as per the manufacturer’s instructions, and using the 14 cycle option and a subsequent 20x dilution into nuclease free water (am9937, Fisher). All RT-qPCR reactions were performed using SSO Universal SYBR Green SuperMix, as per manufacturer instructions (1725275, BioRad). Primer sequences used were TurboGFP (5’TGA TGG GCT ACG GCT TCT A, 5’GTG TTG CTG TGA TCC TCC TC). All RT-qPCR analyses were performed on the StepOnePlus Real Time PCR system (437660, Thermo).

Rosàs-Canyelles E., Modzelewski A.J., Martinez A.E., Geldert A., Gopal A., He L, & Herr A.E. (2021). Multimodal detection of protein isoforms and nucleic acids from low starting cell numbers. Lab on a chip, 21(12), 2427-2436.

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Quantitative analysis of CB1 and CB2 receptor expression

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Total RNAs were isolated from brain regions and peripheral tissues using the TRIzol Reagent. Single strand cDNAs were synthesized using qScript XLT cDNA SuperMix (Quantabio, Beverly, MA, USA, #95161-500). Rodent isoform CB1A [3 (link)] and CB2A [4 (link)] FAM-labeled probes and endogenous control VIC-labeled Actb probe (Thermo Fisher Scientific Inc., Waltham, MA, USA, #4352341E) were used for TaqMan RT-qPCR. To validate hepatocyte CB1R detection, TaqMan PreAmp Master Mix Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA, #4391128) or PerfeCTa PreAmp SuperMix (Quantabio, Beverly, MA, USA, #95146-005) was used for cDNA preamplification using 80 nM of forward and reverse primer sets [48 (link)]. cDNAs were pre-amplified using the program: 95 °C hold for 10 min and then 10 cycles of denaturation at 90 °C for 15 s and annealing and extension at 60 °C for 4 min. Duplex PCR assays containing both the target and endogenous control TaqMan probes were carried out with Advanced TaqMan Fast PCR Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA, #4444556) or PerfeCTa Multiplex qPCR ToughMix (Quantabio, Beverly, MA, USA, #95147-250) in StepOnePlus instrument using a default thermo-cycling program. The relative fold change is calculated using the formula: 2^{(−△△Ct)} [48 (link)].

Liu Q.R., Canseco-Alba A., Liang Y., Ishiguro H, & Onaivi E.S. (2020). Low Basal CB2R in Dopamine Neurons and Microglia Influences Cannabinoid Tetrad Effects. International Journal of Molecular Sciences, 21(24), 9763.

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Verification of miRNA Expression in sEVs

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The miRNAs from the NGS panel were measured in the LI group Pre and Post-24h (n = 6) via qPCR to verify specificity of HI. Following miRNA isolation and purification from sEVs, cDNA was synthesized from miRNA utilizing a qScript miRNA cDNA Synthesis Kit (Cat no. 89168-790; Quantabio, Beverly, MA, USA). Next, cDNA was further enriched using PerfeCTa PreAmp SuperMix (Cat no. 89409-170; Quantabio) to increase cDNA template volume for qPCR application. qPCR assays were then performed using primer sets designed to specifically amplify candidate miRNAs identified through NGS as the most changed in the HI group: miR-92b-5p, miR-423-5p, miR-24-3p, miR-7844-5p, miR-144-5p, miR-221-5p, and miR-22-3p.

Muñoz E.R., Caccese J.B., Wilson B.E., Shuler K.T., Santos F.V., Cabán C.T., Jeka J.J., Langford D, & Hudson M.B. (2020). Effects of purposeful soccer heading on circulating small extracellular vesicle concentration and cargo. Journal of Sport and Health Science, 10(2), 122-130.

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Quantifying mRNA Transcript Abundance

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Each total RNA sample was quantified and analyzed for purity using a spectrophotometer (Nanodrop ND-1000). Five hundred ng of total RNA (from 21EM15 cells or mouse lenses) or 10 ng of total RNA (from explants) were reverse transcribed using the iScript cDNa Synthesis Kit (Bio-Rad, CA) per the manufacturer’s instructions. One ng of cDNA from the explants was then pre-amplified using the Quantabio PerfeCTa PreAmp Supermix (Quantabio, MA) with the same primers as used for qPCR (per the manufacturer’s instructions) and diluted 20-fold. Ten ng of cDNA from the 21EM15 cells, 1 uL of preamplified cDNA from the explants or 75 ng of cDNA from the lenses was then used to estimate the abundance of specific mRNA transcripts using the iTaq Universal SYBR Green Supermix (Bio-Rad, CA) on a CFX96 Real-Time PCR System (Bio-Rad, CA). Relative mRNA transcript abundance was estimated using the ΔCt method [37 (link)] with the housekeeping gene, GAPDH, used as an internal normalization control for each sample. Primer sequences used are provided in Supplemental Table 1.

Thompson B., Davidson E.A., Chen Y., Orlicky D.J., Thompson D.C, & Vasiliou V. (2022). Oxidative stress induces inflammation of lens cells and triggers immune surveillance of ocular tissues. Chemico-biological interactions, 355, 109804.

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Quantitative Real-Time PCR for mRNA Transcripts

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Each total RNA sample was quantified and analyzed for purity using a spectrophotometer (Nanodrop ND-1000). Five hundred ng of total RNA (from 21EM15 cells or mouse lenses) or 10 ng of total RNA (from explants) were reverse transcribed using the iScript cDNa Synthesis Kit (Bio-Rad, CA) per the manufacturer's instructions. One ng of cDNA from the explants was then pre-amplified using the Quantabio PerfeCTa PreAmp Supermix (Quantabio, MA) with the same primers as used for qPCR (per the manufacturer's instructions) and diluted 20-fold. Ten ng of cDNA from the 21EM15 cells, 1 uL of preamplified cDNA from the explants or 75 ng of cDNA from the lenses was then used to estimate the abundance of specific mRNA transcripts using the iTaq Universal SYBR Green Supermix (Bio-Rad, CA) on a CFX96 Real-Time PCR System (Bio-Rad, CA). Relative mRNA transcript abundance was estimated using the ΔCt method [36] with the housekeeping gene, GAPDH, used as an internal normalization control for each sample. Primer sequences used are provided in Supplemental Table 1.

Thompson B., Davidson E.A., Chen Y., Orlicky D.J., Thompson D.C., & Vasiliou V. (2021). Oxidative stress induces inflammation of lens cells and triggers immune surveillance of ocular tissues.

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