exposure: nano_ATZ EC50 (effect concentration on reproduction) in soil for 7 days stage of development: Adult organisms with well developed clitellum
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using SV Total RNA Isolation System (Promega Corporation, USA).The quantity and purity of the RNA samples was checked using the ND-1000 spectrophotometer (Nanodrop®, USA) through measurement of the 260nm/280nm and 260nm/230nm absorbance. For all used samples these ratios were approximately 2. To verify the integrity of the RNA samples, a denaturating formaldehyde agarose gel electrophoresis was performed.
Label
Cy3
Label protocol
Labelling and hybridizations followed the protocol “One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling” from Agilent Technologies (Agilent Technologies, Palo Alto, CA, USA)). Briefly, 500 ng of total RNA was amplified and labelled with Agilent Low Input Quick Amp Labelling Kit (Agilent Technologies, Palo Alto, CA, USA). Positive controls were added with the Agilent one-colour RNA Spike-In Kit (Agilent Technologies, Palo Alto, CA, USA). Purification of the amplified and labelled cRNA was performed with the RNeasy columns (Qiagen, Valencia, CA, USA).
Hybridization protocol
The cRNA samples were hybridized on the Custom Gene Expression Agilent Microarrays (4 x 44k format) developed for Enchytraeus crypticus. Hybridizations were performed using the Agilent Gene Expression Hybridization Kit (Agilent Technologies, Palo Alto, CA, USA) and each biological replicate was individually hybridized on one array. The arrays were hybridized at 65ºC with a rotation of 10 rpm, during 17h. After that the microarrays were washed using Agilent Gene Expression Wash Buffer Kit (Agilent Technologies, Palo Alto, CA, USA)
Scan protocol
Scanning was performed using the Agilent DNA microarray scanner G2505B (Agilent Technologies). Fluorescence intensity data was obtained with Feature Extraction (10.5.1.1) Software (Agilent Technologies).
Data processing
Background correction was provided by Agilent Feature Extraction software and only gene probes with good signal quality (flag IsPosAndSignif = True) in all samples were employed in the analyses.
High-throughput transcriptomics reveals mechanisms of nanopesticides – nanoformulation vs conventional pesticides and active ingredient – a case study with Enchytraeus crypticus
Data table header descriptions
ID_REF
VALUE
processed Cy3 signal intensity (Agilent gProcessedSignal)
