Journal: Cold Spring Harbor protocols

Article Title: Following Endocrine-Disrupting Effects on Gene Expression in Xenopus laevis .

doi: 10.1101/pdb.prot098301

Figure Lengend Snippet: FIGURE 1. (A) Graphical description of treatment. An example is given of a substance to be tested in a starting solution at 10−1 M in DMSO. Targeted dilutions are 10−12, 10−10, 10−8, and 10−6 M. First, a cascade dilution in DMSO is done to obtain a 10,000 times more concentrated solution than the targeted dilution. Then 1 µL of each “mother dilution” is diluted in either 10 mL of Evian water or 5 nM T3 (prepared in Evian water). CTRL or T3 treatments are, respectively, 1 µL DMSO in 10 mL water and 1 µL DMSO in 10 mL 5nM T3. For each concentration tested, 8 mL of the prepared solution (10 mL) are pipetted into the wells and the plates are incubated at 23˚C. Daily renewal is done at a given time. After 72 h, tissues/tadpoles are collected. (B) Quantification of expression of eight typical thyroid hormone-related genes are given as examples obtained in brain tissue after 3-d exposure with T3 5 nM. Genes encoding transcription factors up-regulated in brain tissue after a 3-d exposure with T3 are: thyroid hormone receptor beta (thrb) with two- to threefold induction, kruppel like factor 9 (klf9) with a 10-fold induction, and thyroid hormone induced bzip factor (thibz) with a 20- to 30-fold induction. dio3 (encoding the D3 enzyme which inactivates T3 into T2) is documented to be up-regulated (two- to threefold induction) by T3 while dio1 (encoding D1) is observed to be down-regulated (twofold reduction) and dio2 (encoding the activating enzyme D2 which converts T4 into T3) remains unchanged. Two other genes encoding the nuclear receptor alpha (thra) and the membrane transporter lat2 (lat2) are documented to be down-regulated. These results are the pool of three independent experiments with n = 4 replicates in each experiment. Total RNA extraction for each replicate was obtained from two brains. 500 ng of total RNA was used for reverse transcription and 1:20 dilution cDNA was used for qPCR. See Table 1 for primers. Fold changes are presented as mean + SEM using histograms. Statistics were done using pairwise nonparametric Mann–Whitney test with (****) P < 0.0001, (**) P < 0.01, (*) P < 0.05.

Article Snippet: Evian water in 75 cL glass bottle or another mineral water with reproducible quality High capacity cDNA RT kit (Life technologies 4368813) or Reverse Transcription Master Mix (Fluidigm 100-6299) Human chorionic gonadotropin (hCG; CAS 9002-61-3; Sigma-Aldrich CG5-1VL) Ice Liquid nitrogen Milli-Q reference water Nuclease-free water (ThermoFisher Scientific AM9937) Power SYBR Master Mix (Life Technologies 4368708) Primers, 10 pM (Eurofins; see Table 1) RNAse-free 0.8% agarose gel (optional; see Step 22) RNAqueous–micro kit (Life Technologies AM1931) RnaseZap (ThermoFisher Scientific AM9780; optional; see Step 17) Sodium hydroxide (NaOH 0.1 N; CAS 1310-73-2; optional; see Step 17) Transgenic Xenopus laevis line expressing GFP (e.g., Tg(thibz:GFP) [Fini et al. 2007]) (optional; see Step 36) 3,3′,5-Triiodo-L-thyronine sodium salt (T3) solution Xenopus laevis mature male and female (WT) or Xenopus laevis NF stage 45 tadpoles Equipment 96-well black, conical well plate (Greiner Bio-one 651209; optional; see Step 38) 384-well clear hardshell plate (ThermoFisher Scientific 4309849) Air pump and stone Adhesive covers for 384 well plates (ThermoFisher Scientific 4311971) BioAnalyzer (Agilent) Black foil to cover breeding chamber Microcentrifuge (e.g., Eppendorf 5417 R) Dissecting tools (forceps, etc.)

Techniques: Concentration Assay, Incubation, Expressing, Membrane, RNA Extraction, Reverse Transcription, MANN-WHITNEY