To partially validate the trends of the microarray results, we performed qRT-PCR on the isolated RNA for selected genes with three replicates for each condition and strain (WT, Alg−, and EPS− under −0.4 MPa and −0.5 kPa). The genes and their primer sets are listed in Table S4. cDNA synthesis and amplification were performed using Qscript 1-Step SYBR Green QRT-PCR Kit (Quanta Biosciences, Gaithersburg, MD) in a Chromo4 thermocycler (MJ Research, Waltham, MA) with a total RNA input of 50 ng following the standard protocol as per the manufacturer (Quanta Biosciences). Data were normalized with respect to rimM (coding for the 16S rRNA processing protein RimM), which was not differentially expressed in our study, as done by Li et al. (2010 (link)) and Gulez et al. (2012 (link)). Expression levels of the target genes at −0.4 MPa were calculated relative to levels at −0.5 kPa using the 2▵▵CT method (Livak and Schmittgen 2001 (link)).
Qscript 1 step sybr green qrt pcr kit
Manufactured by Quanta Biosciences
The Qscript 1-Step SYBR Green QRT-PCR Kit is a laboratory instrument used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. It enables the simultaneous reverse transcription and quantitative PCR amplification of RNA targets in a single reaction vessel.
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Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Chromo 4 thermocycler, by Bio-Rad (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Rneasy protect bacteria mini kit, by Qiagen (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Miniopticon, by Bio-Rad (1 mentions)
4 protocols using qscript 1 step sybr green qrt pcr kit
1
Validating Microarray Trends via qRT-PCR
2
Quantitative RT-PCR Analysis of B. cenocepacia
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RNA was isolated from biofilm grown B. cenocepacia strains on membrane filters placed on AB medium supplemented with 2% agar (w/v), 0.2% glucose (w/v), 0.1% casaminoacids (w/v) for 48 hr using the RNeasy Protect Bacteria Mini Kit (Qiagen), and it was DNase treated using the Turbo DNA‐free kit (Ambion) according to manufacturers’ instructions. cDNA synthesis and quantitative RT‐PCR analysis were carried out using the Qscript 1‐Step SYBR green qRT‐PCR kit (Quanta Biosciences) according to manufacturer's instructions. As a control, quantitative RT‐PCR was similarly applied to analyze the expression of the gyrB gene. The relative expression levels of the target genes were calculated using the threshold cycle (2−ΔΔCt) method (Livak & Schmittgen, 2001 ).
3
Quantitative RT-PCR Protocol for Gene Expression
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RT‐PCR was carried out in a single step using the qScript 1‐Step SYBR Green qRT‐PCR kit (Quanta Biosciences, Cummings Center, Beverly, MA), following the manufacturer's instructions. The protocol begins with reverse transcription at 50°C for 10 min for 1 cycle; activation of reverse transcriptase at 95°C for 5 min for 1 cycle; denaturation of samples at 95°C for 10 s, annealing for 20 s, and extension at 72°C for 1 min for 45 cycles. The primers used for each gene and respective amplicon length, concentration, and annealing temperature are described in Table 1 .8 , 27 , 28 , 29 , 30 A LightCycler® 480 thermal cycler (Roche Life Science, PenzbERG, Upper Bavaria, Germany) was used for RT‐qPCR. To obtain the melting curves, the PCR products were heated at 95°C for 5 s, at 65°C for 1 min, at 97°C continuously with 5–10 adq/°C, and then cooled at 40°C for 10 s. UBC was used as a reference expression gene. All tests were carried out in duplicate. All 103 study samples showed similar values of UBC gene expression: cross points (CPs) median = 29.61, mean = 29.68.
4
Placental Gene Expression Quantification
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Tissue was homogenized in buffer RLT using pestles and Qiashredders, and total RNA was extracted using the RNeasy Mini kit (Qiagen, Ontario, Canada). RNA concentration was determined using a Nanodrop spectrophotometer (Thermo Fischer Scientific Inc., Waltham, MA). A reference sample was prepared by combining samples and was included in every assay to account for variation between assays. In addition to the mRNA levels of Hsd11b1 and Hsd11b2, we also measured Slc38a2, Slc27a4, Slc2a1, genes encoding neutral amino acid, long-chain fatty acid and glucose transporters, respectively, expressed in the placenta [23 (link),26 (link),35 (link)] and β-actin as a housekeeping gene. Primer sequences are shown in Table 1 . The qScript 1-step SYBR Green qRT-PCR kit (Quanta Biosciences Inc. Gaithersburg, MD) was used to reverse-transcribe and amplify each sample for 40 cycles. At each cycle, the amount of fluorescence was quantified using a miniOpticon (Bio-Rad, Hercules, CA), and the cycle at which the signal rose above a fixed threshold (Ct) was determined. Each sample was analysed in duplicate. We used the method of Pfaffl [36 (link)] to calculate mRNA expression levels relative to the reference sample, e.g., a value of 1.5 indicates a sample has 50% more of a particular transcript than the reference sample, correcting for β-actin.
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