Preamp master mix

Manufactured by Standard BioTools

Sourced in United States

PreAmp Master Mix is a reagent used in molecular biology workflows. It is designed to facilitate the amplification of target sequences prior to downstream analysis. The core function of this product is to provide the necessary components for efficient pre-amplification of DNA or RNA samples.

Automatically generated - may contain errors

Lab products found in correlation

Reverse transcription master mix, by Standard BioTools (12 mentions) Biomark hd system, by Standard BioTools (11 mentions) Real time pcr analysis software, by Standard BioTools (6 mentions) Evagreen supermix, by Bio-Rad (5 mentions) Biomark hd, by Standard BioTools (5 mentions) Nucleospin rna xs kit, by Macherey-Nagel (4 mentions) Anti cd3 clone okt3, by Thermo Fisher Scientific (3 mentions) Iscript cdna synthesis kit, by Bio-Rad (3 mentions) Ssofast evagreen low rox kit, by Bio-Rad (3 mentions) Rneasy mini kit, by Qiagen (3 mentions) Exonuclease 1, by New England Biolabs (3 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions) Fluidigm reverse transcription master mix, by Standard BioTools (3 mentions) Direct zol rna microprep kit, by Zymo Research (3 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Cfx96 thermocycler, by Bio-Rad (2 mentions) Rneasy plus mini kit, by Qiagen (2 mentions) 96.96 dynamic array ifc, by Standard BioTools (2 mentions) 48.48 dynamic array, by Standard BioTools (2 mentions) Picopure rna isolation kit, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Fluidigm PreAmp Master Mix (8 citations) TaqMan PreAmp Master Mix (4 citations) PreAmp Master Mix kit (4 citations) Perfect PreAmp Master Mix (2 citations) PreAmp and Reverse Transcription Master Mix Kit (2 citations) PreAmp and Reverse Transcription Master Mix (2 citations)

54 protocols using preamp master mix

Activated CD8+ T Cell Gene Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were activated for 5 hr with plate-bound anti-CD3 (clone OKT3; Thermo Fisher Scientific). RNA was extracted from flow-sorted naive CD8⁺ T cells (n = 300 per condition) using a NucleoSpin RNA XS Kit (Macherey-Nagel), and cDNA was synthesized using Reverse Transcription Master Mix (Fluidigm). Specific targets were amplified using PreAmp Master Mix (Fluidigm). Gene expression was assessed using a BioMark HD System (Fluidigm) with EvaGreen Supermix (Bio-Rad). RNA expression levels were calculated using the 2^−ΔΔCT method with reference to a housekeeping gene (human 18S) (18 (link)).

Nicoli F., Cabral-Piccin M.P., Papagno L., Gallerani E., Fusaro M., Folcher V., Dubois M., Clave E., Vallet H., Frere J.J., Gostick E., Llewellyn-Lacey S., Price D.A., Toubert A., Dupré L., Boddaert J., Caputo A., Gavioli R, & Appay V. (2022). Altered basal lipid metabolism underlies the functional impairment of naive CD8+ T cells in elderly humans. Journal of immunology (Baltimore, Md. : 1950), 208(3), 562-570.

+ Open protocol

+ Expand

mRNA and Protein Expression Analysis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For mRNA expression, qRT-PCR analysis was performed using predefined TaqMan probes (Supplementary Data 1) chosen from the Thermo Fisher Scientific database (http://www.thermofisher.com). Using the High Capacity cDNA Reverse Transcription kit (Thermofisher), 1.5 µg of total RNA was reverse transcribed in a final volume of 50 µl. qPCR reactions were done using the high throughput BioMark HD system (Fluidigm) following manufacturer’s instructions. Pre-amplifications of 6 ng cDNA were performed using PreAmp Master Mix (Fluidigm) with a primer mix combining each primer used in the present study except the 18S probe due to it very high gene expression level. Expression data (Ct values) were acquired using the Fluidigm Real Time PCR Analysis software. The mean of 5 housekeeping genes (18S, ACTB, CLTC, GAPDH, and TBP) was used for the normalization of expression data. For protein expression, preparation of cells lysates from 30 frozen tumor samples and reverse phase protein array (RPPA) were performed as previously described^{42 (link)}. Array was revealed with the anti-PD-L1 monoclonal antibody (clone E1L3N; Cell Signaling Technology).

Blum Y., Meiller C., Quetel L., Elarouci N., Ayadi M., Tashtanbaeva D., Armenoult L., Montagne F., Tranchant R., Renier A., de Koning L., Copin M.C., Hofman P., Hofman V., Porte H., Le Pimpec-Barthes F., Zucman-Rossi J., Jaurand M.C., de Reyniès A, & Jean D. (2019). Dissecting heterogeneity in malignant pleural mesothelioma through histo-molecular gradients for clinical applications. Nature Communications, 10, 1333.

+ Open protocol

+ Expand

Retinal gene expression analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Retinas and RPE‐eyecups from 1‐month‐old WT, Adipor1^−/−, and Mfrp^rd6animals (n = 6 each) were isolated. Mutant and WT samples for males and females (n = 3 each) were treated and analyzed separately. There was no difference observed between the two genders; therefore, we combined the data for each type of mouse for n = 6. Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. One microgram of RNA was reverse transcribed using an iScript cDNA Synthesis Kit (Bio‐Rad, Hercules, CA). Diluted cDNA (100 ng, 1.25 µL) was used for the preamplification reaction by adding 1 µL of PreAmp Master Mix (Fluidigm, San Francisco, CA), 0.5 µL of a mix of all primers, and water to a final volume of 5 µL. The temperature profile was 95°C for 2 minutes followed by 12 cycles of amplification (95°C for 15 s, and 60°C for 4 min using the Bio‐Rad CFX96 thermocycler). Each preamplification reaction of cDNA was then subjected to Exonuclease I treatment, to remove unincorporated primers. The resulting preamplified and treated cDNA was then diluted 5 times in TE Buffer.

Kautzmann M.I., Gordon W.C., Jun B., Do K.V., Matherne B.J., Fang Z, & Bazan N.G. (2019). Membrane‐type frizzled‐related protein regulates lipidome and transcription for photoreceptor function. The FASEB Journal, 34(1), 912-929.

+ Open protocol

+ Expand

Single-Cell Integrated Mutation and Expression

Cited 3 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

Integrated detection of somatic mutations, SNPs within regions of chromosomal deletion, and gene expression in the same single cell was performed on a Biomark HD instrument (Fluidigm) (Livak et al. 2013 (link); Vargas et al. 2016 (link)). cDNA was generated using reverse transcription master mix (100-6299, Fluidigm), and preamplification was performed with PreAmp master mix (100-5744, Fluidigm) and 10× preamplification primer mix (500 nM each primer). For gene expression, allele-specific mutation and SNP detection, qPCR was performed using 96.96 dynamic array IFCs (Fluidigm) as previously described (Livak et al. 2013 (link); Burger et al. 2016 (link)). Data were analyzed with the Fluidigm real-time PCR analysis software using the linear (derivative) baseline correction method and the auto (global) Ct threshold method. The C_q values determined were exported, and data were processed as the C_q threshold (set to 28) minus the experimental C_q values. The transformation yields values equivalent to a log2 transformation of sequencing read count. For more details on primer design, cDNA generation, and preamplification conditions, please see the Supplemental Methods.

Wang L., Fan J., Francis J.M., Georghiou G., Hergert S., Li S., Gambe R., Zhou C.W., Yang C., Xiao S., Cin P.D., Bowden M., Kotliar D., Shukla S.A., Brown J.R., Neuberg D., Alessi D.R., Zhang C.Z., Kharchenko P.V., Livak K.J, & Wu C.J. (2017). Integrated single-cell genetic and transcriptional analysis suggests novel drivers of chronic lymphocytic leukemia. Genome Research, 27(8), 1300-1311.

+ Open protocol

+ Expand

Multiplex qPCR Analysis of Tumor-Infiltrating CD45+ Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Tumor-infiltrating CD45⁺ cell-derived cDNA was subjected to gene expression analysis using the 96.96 Dynamic Array™ IFC (integrated fluidic circuits) for Gene Expression of the Fluidigm BioMark HD system. Briefly, 1 μl of PreAmp Master Mix (Fluidigm), 0.5 μl of pooled mixed primers (500 nM each, Fluidigm Delta Gene Assays, Table S2), and 2.25 μl of water were added to 1.25 μl of cDNA and 14 cycles of STA (specific target amplification) were performed according to the manufacturer’s recommendations. After the STA, an Exonuclease I digestion was performed to remove unincorporated primers by adding 0.4 μl Exonuclease I (20 U/μL), 0μL Exonuclease I Reaction Buffer and 1.4 μl water to each sample, followed by vortexing, centrifuging and heating the sample to 37°C for 30 minutes. After a 15 minute 80°C heat inactivation, the amplified sample was diluted 1:5 in Buffer TE. To perform the multiplex quantitative PCR run on the Biomark instrument, sample mix (5 μl) and assay mix (5 μl) was prepared and loaded onto the 96.96 Dynamic Array IFC (integrated fluidic circuits), following the program instructed by the manufacturer. Standard EvaGreen based Quantitative Real-time PCR (qPCR) was performed using primers listed in Table S3.

Liu C., Chikina M., Deshpande R., Menk A.V., Wang T., Tabib T., Brunazzi E.A., Vignali K.M., Sun M., Stolz D.B., Lafyatis R.A., Chen W., Delgoffe G.M., Workman C.J., Wendell S.G, & Vignali D.A. (2019). Treg cells promote the SREBP1-dependent metabolic fitness of tumor-promoting macrophages via repression of CD8+ T cell-derived interferon-γ. Immunity, 51(2), 381-397.e6.

+ Open protocol

+ Expand

Validating RNA-seq Differential Expression

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

16 RNA samples were obtained by the procedures, described above, from another batch of female Brd1^+/− and WT mice (8/group). 180 ng total RNA was reverse transcribed by iScript Select cDNA Synthesis Kit (Bio-Rad, Hercules, USA). All eight DEGs, detected by DESeq2 and TSPM, were selected for validation. 107 more genes were randomly selected for validation from the list of remaining 191 DEGs, detected by Cuffdiff2 and edgeR. After 10–20 cycles of specific target amplification with PreAmp master mix (Fluidigm, San Francisco, USA), high-throughput qPCR was performed on the BioMark HD (Fluidigm, San Francisco, USA), using 48.48 dynamic arrays (Fluidigm, San Francisco, USA) and SsoFast EvaGreen Low ROX kit (Bio-Rad, Hercules, USA) [Additional files 12 & 13]. A DEG, detected by the RNA-seq DEG analysis methods, was considered as a true-positive DEG, if it satisfied the following criteria, (i) Both RNA-seq and qPCR showed same direction (upregulation or down-regulation) of differential expression, (ii) Differential expression fold change, estimated by qPCR, was either above 1.25 or below 0.80 (LFC cut-off was ±0.3219) [34 (link)]. Spearman correlation coefficients, root-mean-square deviations and kappa statistics were calculated using STATA 13.1 (StataCorp LP, Texas, USA).

Rajkumar A.P., Qvist P., Lazarus R., Lescai F., Ju J., Nyegaard M., Mors O., Børglum A.D., Li Q, & Christensen J.H. (2015). Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq. BMC Genomics, 16(1), 548.

+ Open protocol

+ Expand

Quantitative Analysis of c-myc mRNA Levels

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA from IP samples spiked with drosophila mRNA was isolated using RNeasy Mini Kit (74104, Qiagen). First-strand synthesis was performed using M-MLV Reverse Transcriptase (2805013, Thermo Fisher Scientific) and oligo(dT). cDNA was preamplified using PreAmp Master Mix (1005580, Fluidigm) following manufacturer’s instructions. Quantitative amplification was performed using SYBR Green PCR Master Mix in an 7900HT thermocycler (Applied Biosystems). mRNA level were detected using the following primer sequences: c-myc fwd TTT GTC TAT TTG GGG ACA GTG TT, c-myc rev-CAT CGT GGC TGT CTG, dro-rpl32 fwd-ATG CTA AGC TGT CGC ACA AAT G, dro-rpl32 rev-GTT CGA TCC GTA ACC GAT GT. mRNA level were normalized to drosophila rpl32 mRNA level.

Liedmann S., Liu X., Guy C.S., Crawford J.C., Rodriguez D.A., Kuzuoğlu-Öztürk D., Guo A., Verbist K.C., Temirov J., Chen M.J., Ruggero D., Zhang H., Thomas P.G, & Green D.R. (2022). Localization of a TORC1-eIF4F translation complex during CD8+ T cell activation drives divergent cell fate. Molecular cell, 82(13), 2401-2414.e9.

+ Open protocol

+ Expand

Reverse Transcription and Pre-amplification Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

cDNA was prepared from the extracted RNA by reverse transcription using 5 µL Reverse Transcription Master mix (Fluidigm, California, USA). The resulted cDNA was then pre-amplified by using PreAmp Master mix (Fluidigm) with 7 custom-designed primers (designed by using Delta Gene assays according to the manufacturer's instructions). The pre-amplification reactions were performed with BioRad MyCycler Thermal Cycler (Bio-Rad Laboratories, Inc.) using the following amplification conditions: 95 °C for 2 min, followed by 14 cycles of 95 °C for 15 s and 60 °C for 4 min.

Nakkarach A., Foo H.L., Song A.A., Mutalib N.E., Nitisinprasert S, & Withayagiat U. (2021). Anti-cancer and anti-inflammatory effects elicited by short chain fatty acids produced by Escherichia coli isolated from healthy human gut microbiota. Microbial Cell Factories, 20, 36.

+ Open protocol

+ Expand

miRNA Quantification Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Preamplification was performed using miRCURY LNA miRNA PCR Assay primers (Qiagen, France, Cat. 339306) for the 21 miRNAs plus RT UniSp6 Spike-in and Extraction Spike-in cel-miR-39. The product of reverse transcription was purified with Exonuclease I (New England Biolabs France, Paris, France, Cat. M0293).
The product of Reverse Transcription was then diluted 1/10, and 1.25 µL of diluted cDNA was preamplified using Fluidigm PreAmp Master mix (Paris, France, Cat. 100-5580) and primer mix in 5 µL reaction volume according to the supplier’s specifications.

Brandão-Lima P.N., de Carvalho G.B., Payolla T.B., Sarti F.M., Fisberg R.M., Malcomson F.C., Mathers J.C, & Rogero M.M. (2022). Circulating microRNAs Showed Specific Responses according to Metabolic Syndrome Components and Sex of Adults from a Population-Based Study. Metabolites, 13(1), 2.

+ Open protocol

+ Expand

Fluidigm Pre-Amplification Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Pre-amplification was performed according to Fluidigm instruction manual: for every nano-fluidic chip, a pooled primer mix was prepared by adding 1 μL of primer stock (for every target gene to be tested on the chip) to water up to a final volume of 100 μL. Every primer stock contained both reverse and forward primers at a concentration of 50 μM each. A pre-amplification mix was prepared containing for each sample: 1 μL of PreAmp Master mix (Fluidigm PN 100–5744), 0.5 μL of pooled primer mix and 2.25 μL of nuclease free water. 3.75 μL of pre-amplification mix was then aliquoted in a 96 well-plate. 1.25 μL of cDNA was then added in each well. The samples were mixed by quick vortexing and centrifuged. Pre amplification conditions were the following: 95ºC for 2 min, 10 cycles of denaturation at 95ºC for 15 s followed by annealing/extension at 60ºC for 4 min.

Haeussler S., Köhler F., Witting M., Premm M.F., Rolland S.G., Fischer C., Chauve L., Casanueva O, & Conradt B. (2020). Autophagy compensates for defects in mitochondrial dynamics. PLoS Genetics, 16(3), e1008638.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!