Droplet digital PCR (ddPCR) of hydrazine synthase (hzsB), ammonia monoxygenase (amoA), nosZ clade I genes, and transcripts used the QX200 platform with 20 µl reactions, 10 µl 2× EvaGreen Supermix (Biorad, Hercules, CA, US), and 1 µl DNA or cDNA. RNA (24 pg–1.1 µg) was converted to cDNA using Superscript III Supermix (Invitrogen). DPEC-treated water was used for negative controls and gBlock dsDNA fragments were used as positive controls (Table S2 ; Integrated DNA Technologies, Coralville, IA, USA). Primers and PCR conditions are described in Table S2 . Data were analyzed using the QuantaSoft software package v1.0 (Bio-Rad). Positive droplet thresholds were set based on negative and positive droplet fluorescence amplitudes using the positive control as reference.
Evagreen supermix
Manufactured by Bio-Rad
Sourced in United States, Canada
EvaGreen Supermix is a ready-to-use qPCR master mix that contains the fluorescent dye EvaGreen for real-time detection of DNA amplification. It is designed for sensitive and specific quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (15 mentions)
Qx200 droplet reader, by Bio-Rad (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (7 mentions)
Biomark hd system, by Standard BioTools (6 mentions)
Iscript cdna synthesis kit, by Bio-Rad (6 mentions)
Quantasoft software, by Bio-Rad (6 mentions)
Maxima h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Preamp master mix, by Standard BioTools (5 mentions)
Qx200, by Bio-Rad (5 mentions)
Qx200 droplet generator, by Bio-Rad (5 mentions)
Ddpcr, by Bio-Rad (4 mentions)
C1000 touch thermal cycler, by Bio-Rad (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Qx200 system, by Bio-Rad (3 mentions)
Anti cd3 clone okt3, by Thermo Fisher Scientific (3 mentions)
Reverse transcription master mix, by Standard BioTools (3 mentions)
Nucleospin rna xs kit, by Macherey-Nagel (3 mentions)
Cfx96 real time system, by Bio-Rad (3 mentions)
Superscript vilo reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
113 protocols using evagreen supermix
1
Quantitative Microbial Gene Expression Analysis
2
Droplet Digital PCR Quantification Protocol
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ddPCR was performed by mixing 11 µl of 2× EvaGreen Supermix (Bio-Rad), 1 µl of 2 µM stocks for each primer (see Table 2 ), and distilled water (dH2O) to reach a total volume of 20 µl for each reaction. The mix was dispersed into PCR strips, and then 2 µl of the template was added. The reaction mixtures were loaded into a QX200 system (Bio-Rad) for droplet generation. Droplet generation required 20 µl of sample and 70 µl of droplet generation oil for EvaGreen (Bio-Rad). Droplets were created in a volume of 40 µl and were transferred to a High-Profile 96-well PCR plate (Bio-Rad). The plate was sealed using a PX1 PCR plate sealer (Bio-Rad) and put into a thermocycler with a ramp rate of 2°C/s. The cycling program was 95°C for 10 min, 40 cycles of 95°C for 30 s and 60°C for 1 min, and then 4°C for 5 min and 90°C for 5 min, followed by an optional infinite hold at 4°C. After amplification, droplet signals were analyzed using a QX200 reader (Bio-Rad). Data were analyzed using QuantaSoft Analysis Pro with the default setup. Only results from wells where RNA samples were partitioned in >10,000 droplets were included in further calculations.
3
Droplet Digital PCR for Copy Number Variation
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A Droplet Digital PCR (ddPCR) QX200 system (Bio-Rad Laboratories, Hercules, CA, USA), was used to perform ddPCR to confirm copy number change. PCR primers amplifying all three candidate loci and reference primers within the GNAS and RPPH1 genes were designed using Primer3. Ten nanograms of genomic DNA and 2 μM primer pair were added to the 20 μl PCR mixture containing 10 μl 2×EvaGreen Supermix (Bio-Rad). Subsequently, 70 μl Droplet Generator Oil for EvaGreen (Bio-Rad) was added and droplet generation was performed using a DX200 Droplet Generator following manufacturer’s protocol. The micro-reactor emulsion was thermal cycled 40 times under the following conditions after initial denaturation at 95 °C for 30 s: denaturation 95 °C for 30 s, annealing and extension 62 °C for 2 min, signal stabilization 4 °C for 5 min, then 90 °C for 5 min. Amplified droplet number was counted using a QX200 Droplet Reader (Bio-Rad). Absolute droplet numbers of RPPH1 gene were used to calculate the copy number of three candidate loci with GNAS gene as control. Absolute droplet numbers of target loci were divided by that of RPPH1 gene, then multiplied by two to calculate the relative copy number.
4
Activated CD8+ T Cell Gene Expression
5
Absolute Quantification of BDNF-AS and miR-125a/b-5p
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DDPCR was performed to quantify the absolute RNA copy numbers of BDNF-AS and miR-125a/b-5p in MM1S and U266 cells. In brief, 1 μg total RNA samples were reversely transcribed using a First-Strand cDNA Synthesis Kit. The droplets were generated using the QX200™ AutoDG™ Droplet Digital™ PCR System. The PCR reactions using the droplets were prepared using EvaGreen Supermix (1,864,033, Bio-Rad) containing 1 μl of cDNA. The absolute RNA copy numbers were assessed using QX200 Droplet Digital PCR System and calculated as previously described [37 (link)]. The copy numbers per cell were further estimated using a reference mRNA of known abundance as previously described [38 (link)].
6
Dissecting MRE11 Function in DSB Response
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HeLa cells (ATCC) were grown under standard tissue culture conditions (37°C, 5% CO2) in MEM+Glutamax (GIBCO), 10% FBS, 1% non-essential amino acids and 1% sodium pyruvate. Cells were transfected with siRNA (Sigma) against 3′ UTR of MRE11 or luciferase as control (Table S1 ) in complete media for 48 h with the standard protocol for RNAimax. These cells were then transfected with 1 μg of either mammalian ER-I-PpoI-expressing plasmid (Berkovich et. al., 2007 (link)) or empty vector with the standard protocol for Lipofectamine 2000 (Invitrogen). In the experiment where mutants of MRE11 were expressed, pICE-HA-MRE11-WT, pICE-HA-MRE11-H129N, pICE-HA-MRE11-H63D and pICE-HA-MRE11- H63S (Addgene, Chanut et al., 2016 (link)) were co-transfected. The knockdowns and expression of MRE11 mutants was confirmed by immunoblotting. The nuclear translocation of I-PpoI was activated 24 h later by supplementing the media with 4-OHT, 2 μM for 3 h. Cells were harvested, washed and total RNA was extracted using Maxwell® RSC simplyRNA Blood Kit (Promega). RNA was quantified using Nanodrop (Thermoscientific) and 1 μg of total RNA was reverse transcribed using Superscript First strand cDNA synthesis kit with strand-specific primers (Table S1 ). RT-qPCR was used to determine the expression of DSB-induced transcripts using Evagreen supermix (Bio-Rad).
7
Absolute Gene Expression Analysis by ddPCR
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RNA extraction from the liver of juveniles (n = 9 from each experimental group) was carried out using a similar protocol described above for the qPCR. Absolute gene expression analysis was performed with Droplet Digital PCR (ddPCR) using Bio-rad QX200 (Hercules, CA, USA) systems, using the same cDNA obtained as above. Samples were prepared using the workflow provided by the manufacturer. In summary, for each gene, master mixes were prepared using 10 μL EvaGreen super mix (Bio-rad, Hercules, CA, USA), 0.2 μL F primer, 0.2 μL R primer, 7.6 μL MilQ water, and 2 μL cDNA (approx. 20 ng cDNA). Primers, Genbank accession numbers and reference articles for sequences of target (lpl, ppara, elovl6, fads2, cox2, cpt1) and housekeeping gene (ß-act) were provided in Supplementary Table S1 . Droplets were generated using droplet generator Bio-rad QX200 (Hercules, CA, USA) and were transferred to 96-well microplates for PCR in a thermal cycler (Bio-rad C1000 Touch, Hercules, CA, USA). Following PCR amplification, droplets were read in a droplet reader (Bio-rad QX200, Hercules, CA, USA) to determine absolute gene expression. Readings lower than 12000 droplets were not used for the gene expression.
8
RNA Extraction and qPCR Analysis
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Perfused left lungs or cell monolayers were homogenized in TRIzol (Invitrogen, Carlsbad, CA) and total RNA was extracted using a chloroform extraction method as previously described (27 (link)). After DNAse treatment (Invitrogen), cDNA was generated using the Taqman Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA). Samples were quantified on AriaMx real-time qPCR system (Agilent Technologies, Santa Clara, CA) using EvaGreen supermix (Bio-Rad, Hercules, CA) and primers as described (Integrated DNA Technologies, Coralville, IA) (Table S1 https://lungmap.net ).
9
Absolute Gene Expression Analysis of fads2 in PBCs and Liver
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Total RNA extraction from peripheral blood cells and liver were made using a similar protocol described above for the qPCR. Absolute gene expression of PBCs and liver fads2 analysis were performed using Digital Droplet PCR (ddPCR) (Bio-rad QX200, Hercules, CA, USA) systems, by using cDNA obtained as mentioned in Section 2.3 . Sample preparation for ddPCR was carried out as per the manufacturer’s protocol. The master mixes for fads2 gene were prepared including 10 μL EvaGreen super mix (Bio-rad, Hercules, CA, USA), 0.2 μL F primer (10 pmol/μL), 0.2 μL R primer (10 pmol/μL), 7.6 μL MilQ water, and 2 μL cDNA. Then, droplets were generated using droplet generator Bio-rad QX200 (Hercules, CA, USA) and the droplets were transferred to 96 well microplates for PCR in a thermal cycler (Bio-rad C1000 Touch, Hercules, CA, USA). After PCR amplifications, droplets were measured with a droplet reader (Bio-rad QX200, Hercules, CA, USA) to determine absolute gene expression of fads2 gene. The samples with less than 12,000 droplets were not used for the gene expression study. The fads2 gene expression analysis was performed in two replicates for each sample and values were expressed as mRNA copies/µL [31 (link),33 (link)].
10
Quantifying CB2 Receptor Expression
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