Nucleospin 8 rna kit

Tissue homogenization was achieved using the Precellys 24 tissue homogenizer: 2 × 30 s at 6000 rpm, and RNA extraction was performed using the Nucleo Spin 8 RNA kit (Machery-Nagel GmbH & Co. KG, Düren, Germany, Cat. No. 740465.4) with vacuum extraction. For the diet samples we used four biological replicates, using 10 pooled larvae per time point. Samples after early exposure to environmental chemicals were taken from two independent exposure experiments. From each experiment, 3 biological replicates containing 10 pooled larvae were sampled. After RNA extraction, total RNA concentration was measured using Nanodrop (ND-1000, NanoDrop Technologies, Inc., Wilmington, DE, USA) spectrophotometer. One µg of RNA was used for first-strand cDNA synthesis using the high-capapity cDNA synthesis kit (Thermo Fischer, Applied Biosystems^TM, Foster City, CA, USA, Cat. No.: 4368814), and contained 8 mM dNTPs, random hexamers, 5 U/µL reverse transcriptase and 1.0 µg total RNA. Reaction conditions were: 10 min 25 °C, 120 min 37 °C and 5 min 85 °C.

RNA from snap-frozen kidney cortex samples (∼15 mg per animal) stored at −80°C was extracted using a NucleoSpin 8 RNA kit (Macherey-Nagel) and a vacuum manifold. The RNA quantity was measured using a NanoDrop, and RNA quality was determined using an Agilent Bioanalyzer with an RNA 6000 Nano Kit (5067-1511, Agilent, Santa Clara, CA, USA).

Total RNA was extracted from 100 mg leaf or root material using Nucleospin® 8 RNA kit following the manufacturer's protocol (Macherey-Nagel). The quality of RNA was checked in 4200 Tapestation (Agilent Technologies), followed by cDNA synthesis using iScript™ Reverse Transcription Supermix (Bio-Rad). The cDNA samples were used to study gene expression by quantitative real-time PCR (CFX384 Touch™, Bio-Rad) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). The thermal cycling protocol included a single polymerase activation step at 98°C for 3 min followed by 40 amplification cycles, a final extension step at 72°C for 5 min as well as melt-curve analysis. Each amplification cycle comprised a denaturation step at 98°C for 15 s, a primer annealing step at 60/61°C for 30 s and a brief extension at 72°C for 15 s. Primer efficiency was determined by performing standard curve analysis. All qPCR expression data were obtained from CFX Manager Software Version 3.1 (Bio-Rad). The expression of candidate genes was normalized against GAPDH, Cyclophilin (CYC), ACTIN and ADP-ribosylation factor (ADP-RF1). Primers for all candidate genes were designed using Primer3 software and listed in Supplemental Table 1.

Total RNA was extracted from each tissue by homogenization in Trizol reagent (Invitrogen, California, USA) and purified using a silica-membrane technology under vacuum (Nucleospin® 8 RNA kit, Macherey-Nagel, France). The method included a DNase digestion step to remove contaminating DNA. The quantification of RNA was performed by using a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Illkirch, France). Ratios of A260/280 and A260/230 were greater than 1.7 in all samples, denoting good purity. The RNA integrity was assessed using the Agilent RNA 6000 Nano kit and an Agilent 2100 Bioanalyzer (Agilent Technologies France, Massy, France). Average RNA integrity numbers were 8.4 ± 0.4 (mean ± SD). Altogether, 192 RNA samples (n = 48 per tissue; 4 tissues) were thus generated for further analyses.

Total RNA from the kidney cortex was isolated using the NucleoSpin 8 RNA Kit (Macherey–Nagel, Düren, Germany). Possible DNA contamination was eliminated using the RNase-Free DNase Set (QIAGEN, Hilden, Germany), and 1 µg total RNA was reverse transcribed into cDNA with the Reverse Transcription System (Promega, Madison, WI, United States). Gene expression was assessed by semi-quantitative real-time PCR by LightCycler FastStart DNA Master SYBR Green 1 (Roche Diagnostics, Mannheim, Germany) using a thermocycler (qTOWER², Analytik Jena, Germany). PCRs were carried out with sense and antisense primers at a concentration of 0.25 µM each (TIB MOLBIOL, Berlin, Germany). Temperatures and sequences of all primer pairs are shown in Table 1. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as the housekeeping gene. The relative expression ratio was quantified by the ∆∆CT method, and the transcript levels were normalized to the mean value of the young male wild-type control group.

For Arabidopsis, the extraction of total RNA from roots or shoots and the RT-qPCR experiments were performed as previously described [50 (link)]. RNAseq experiments were performed as previously described [43 (link)].
For plant crops, Nucleospin 8 RNA kit (Macherey–Nagel) was used to isolate the RNA from the different plant species tested (50 mg root powder/sample). The quality and concentration of all samples were checked using 4200 Tapestation (Agilent Technologies), followed by DNase treatment and cDNA synthesis from 1 µg of RNA (iScriptTM gDNA). RT-qPCR reactions were performed in a Real-Time PCR Detection System (BIO-RAD) using a total of 10 µl reaction containing 5 µL of Universal SYBR Green Supermix (BIO-RAD) and primers at 0.5 µM. All reactions were performed in technical triplicates. Primers were designed with Primer3. The list of all primers used is provided in Table S3. Relative expression changes were calculated by the ΔCCq method. The exponential expression is calculated as 2^−ΔCq, in which ΔCq is the difference between the Cq of the phosphate starvation induced gene analyzed and the average of Cq obtained from all housekeeping genes used. Values were then normalized to corresponding control.