To assess gene expression levels, synchronized young adult worms were grown and frozen in liquid nitrogen. RNA extraction was performed according to a previously published method, with minor modifications [49 (link)]. Briefly, samples were collected and immediately snap frozen in liquid nitrogen. The following steps were performed after the samples were stored at −80 °C. Samples were resuspended in 500 μl of Trizol (catalog no. 10296010, Thermo Fisher). After 3 freeze–thaw cycles, 100 μl of chloroform (Merck, catalog no. C2432) was added. After centrifugation, the upper phase was collected and mixed with the same volume of 70% ethanol. The RNeasy Plus Mini Kit (Qiagen, catalog no. 74134) was used for further total RNA isolation according to the manufacturer’s instructions. Total RNA (500 ng) was used to generate cDNA using the SuperScript IV First-Strand Synthesis System (Invitrogen, catalog no. 18091050, Thermo Fisher). RT-qPCR was performed using SensiFAST SYBR Hi-ROX mix (2x; Bioline, catalog no. BIO-92020) in a LightCycler480 (Roche) with a 96-well white plate (Roche, catalog no. 4729692001). Fold changes in mRNA expression of the target genes were calculated using the ΔΔCt method. The mean expression levels of act-1 and cdc-42 were used as internal controls. The data are presented as mean ± SD (n = 3). The primers that were used in this study are shown in S5 Table .
96 well white plate
Manufactured by Roche
The 96-well white plate is a laboratory equipment designed for use in various assays and experiments. It features a 96-well format, with each well being white in color. The primary function of this plate is to provide a standardized and consistent platform for conducting experiments, analysis, and data collection.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Sensifast sybr hi rox mix, by Meridian Bioscience (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Chloroform, by Merck Group (1 mentions)
Sypro orange, by Roche (1 mentions)
Sypro orange, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
2 protocols using 96 well white plate
1
Gene Expression Analysis in Synchronized C. elegans
2
Thermal Shift Assay for PrP Interactions
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Fluorescence-based thermal shift assay was used to measure interactions between recombinant human PrP and compounds, which were revealed as temperature changes. These changes were detected using a LightCycler® 480 system (Roche Diagnostics) and SYPRO orange (Life Technologies), which has a maximum excitation at 470 nm and maximum emission at 570 nm. The reaction mixture was as follows: 100 μg/mL recombinant PrP, 150 mM NaCl, 25 mM PIPES (pH 7.0) and 1000-fold diluted SYPRO orange with or without 10 μM compounds in a 96-well white plate (Roche Diagnostics). PrP stabilisation was evaluated by measuring optical density at each temperature. The temperature was gradually increased from 37 °C to 90 °C and fluorescence scanning was continuously measured in 1-°C increments using the LightCycler® 480 instrument with a wavelength filter of 483/610 nm. The fluorescence intensity from each protein dissociation curve was plotted for each thermal point and fitted to the Boltzmann sigmoidal equation using Origin 8.5 software. Each Tm (melting temperature) value was calculated from the maximum value of the first derivative curve of the melting curve, respectively. The mean ± standard deviation (SD) obtained from results of at least three experiments was considered for data analysis.
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