We used the bimake.com 2 × SYBR Green qPCR Master Mix. Four ng cDNA was used for each 20 ul reaction. Three technical replicates were assayed for each sample. Samples were run on a standard 2-step amplification program on Applied Biosystems MicroAmp Fast Optical 96-Well or MicroAmp Optical 384-Well reaction plates on an Applied Biosystems StepOne Plus or ViiA 7, respectively. Primer sequences used either had been previously validated in the Blackshaw lab or were taken from the Harvard Primer Bank or generated using Primer340 (link),49 (link). Those generated on Primer3 were required to span an exon-exon junction. All primers were verified to produce a single reaction product with traditional PCR before use for qPCR. All plots generated with ggplot2 R package50 (link).
Microamp fast optical 96 well
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MicroAmp Fast Optical 96-well is a laboratory equipment used for sample processing and analysis. It provides a 96-well format for high-throughput applications.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Viia7, by Thermo Fisher Scientific (1 mentions)
Sybr green qpcr master mix, by Selleck Chemicals (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna, by Macherey-Nagel (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Microamp optical adhesive film, by Thermo Fisher Scientific (1 mentions)
Perfecta sybr green fastmix low rox, by Quanta Biosciences (1 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
4 protocols using microamp fast optical 96 well
1
Optimized qPCR Assay for Gene Expression
2
Colon RNA Extraction and qPCR Analysis
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RNA was extracted from the colon samples using the NucleoSpin RNA® protocol (Macherey-Nagel, Düren, Germany). For each sample, mRNA (in µg/µL) was measured with a spectrophotometer (Nanodrop 1000; Thermo Scientific). The ratio of each of our samples was >2.0, which was a sign of pure RNA. The cDNA synthesis was performed using a high-capacity DNAc reverse-transcription (RT) master mix (Applied Biosystems, Waltham, MA, USA). For a final volume of 10 µL/sample, the mix contained 2 µL 10X RT buffer, 2 µL 10X RT random primers, 0.8 µL 25X dNTP mix, 1 µL reverse transcriptase, and 4.2 µL RNase-free H2O. PCR was performed in a 96-well microplate (MicroAmp Fast Optical 96-well; Applied Biosystems). For a final volume of 12 µL/well of each sample, a mix was made containing 0.25 µL of forward and reverse primer, 6 µL of SYBR green (Applied Biosystems), 3 µL of RNase-free H2O, and 2.5 µL of cDNA from the samples. SYBR green dye intensity was determined using one-step software. The used primers are provided in Table S2 in the Supplementary Materials . The mRNA levels were normalized to the reference gene (POLR2A (mRNA)) and are reported as fold-change in expression over the control group (CTL).
3
Quantifying Bacterial DNA in Air Samples
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qPCR on extracted DNA from area air samples was performed in a QuantStudio 7 Flex Real-Time PCR System using MicroAmp Fast Optical 96-well and 384-well reaction plates (0.1ml), MicroAmp optical adhesive film, (all Applied Biosystems) and PerfeCTa SYBR Green FastMix, Low Rox (Quanta Biosciences). Each reaction contained 10ul of 2X Master Mix, 4 ul of DNA template, 625 nM of each primer (IDT), and PCR grade water to a final volume of 20 ul. Amplification was comprised of a 10-minute activation step at 95°C, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and a fluorescence measurement. Melting curve analysis was done by monitoring fluorescence throughout incremental increases of temperature from 60°C to 95°C.
The qPCR primers (1369F-1492R) [22 (link)] target regions flanking V9 of the 16S rRNA gene. The standard curve was made using a serially diluted plasmid that contains nt 1369 to 1492 of an E. coli 16S rRNA gene. The concentrations of unknowns are calculated from CT values using the equation generated from plotting the standard curve. All samples are run in triplicate, including the standard curve, a set of non-template controls (NTC), and inhibitor controls (known positives + unknown DNA).
The qPCR primers (1369F-1492R) [22 (link)] target regions flanking V9 of the 16S rRNA gene. The standard curve was made using a serially diluted plasmid that contains nt 1369 to 1492 of an E. coli 16S rRNA gene. The concentrations of unknowns are calculated from CT values using the equation generated from plotting the standard curve. All samples are run in triplicate, including the standard curve, a set of non-template controls (NTC), and inhibitor controls (known positives + unknown DNA).
4
Optimized PCR Primer Annealing Temperatures
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The functioning and optimal annealing temperature of the PCR primers was first examined with gradient PCR (BioRad thermal cycler, USA). The PCR conditions that follow were applied: an initial DNA denaturation at 95 °C for 20 s, primer annealing at 55-65 °C for 20 s, and primer extension at 72 °C for 45 s. This was completed with a final extension step at 72 °C for 5 min. Quantitative PCR was carried out using a StepOnePlus Real-Time PCR System (Applied Biosystems, USA). All PCRs were conducted in duplicate with a volume of 20 µL, using 96-well optical-grade PCR plates and an optical sealing tape (MicroAmp Fast Optical 96-well, Applied Biosystems, USA). EvaGrean dye was used to detect dsDNA synthesis. The thermal cycling conditions included an initial DNA denaturation step at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, primer annealing at the optimal temperature for 20 s, and extension at 72 °C for 30 s. Eventually, melt-curve analysis was done by slow heating of the PCRs to 95 °C (0.3 per cycle) with concurrent measurement of the EvaGrean signal intensity. To demonstrate the absence of contamination or primer dimmer, a non-template control (NTC) reaction with each primer was conducted on gel electrophoresis (Fig. 1 ).
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