Chromo4 system

Two hours into the 12-hour dark cycle, mice were injected intraperitoneally (10 mL/kg) under dim red light. Mice were dosed with vehicle or 15 mg/kg flibanserin 48 hours, 24 hours and immediately prior to a one-hour bright light exposure, and 24 and 48 hours after light exposure. Retinas from vehicle-injected mice and flibanserin-injected mice were harvested 48 hours and 72 hours after light exposure. Total RNA was extracted from retinas using an RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was converted to cDNA using a Bio-Rad iScript cDNA synthesis kit (Hercules, CA). Primers were designed with the Integrated DNA Technologies Realtime PCR Tool (S1 Table, Coralville, Iowa). PCR reaction mixtures consisted of 10 μL DyNAmo HS SYBR green (Thermo Scientific, Waltham, MA), 1 μL cDNA template, 1 μL of 200 μM gene-specific primer sets, and 8 μL distilled water. Using a Bio-Rad Chromo4 System (Hercules, CA), samples were initially denatured for 2 minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds and 58°C for 30 seconds. Gene expression was normalized to β-actin and relative gene expression in flibanserin-injected mice was compared to vehicle-injected mice using the ΔΔCt method.

Community DNA was extracted using Yu and Morrison (36 (link)) method. The DNA was purified using columns from QIAgen DNA Mini Stool Kit (QIAGEN, Valencia, CA, USA). Standard PCR conditions for Fibrobacter succinogenes were as follows: 30 s at 94°C for denaturing, 30 s at 60°C for annealing, and 30 s at 72°C for extension (48 cycles). For the first cycle, 9-min denaturation time was used. In the last cycle, extension was for 10 min. Amplification of 16S rRNA for Ruminococcus flavefaciens and Ruminococcus albus was defined in a similar method, with the exception that the annealing temperature was set to 55°C according to Koike and Kobayashi (38 (link)). The isolation of genomic DNA was used in real-time quantitative PCR assays with power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), forward and reverse primers, and template DNA. Specified primers were used to measure the microbial population of total bacteria according to Edwards et al. (39 (link)), F. succinogenes, R. flavefaciens, and R. albus (38 (link)), Butyrivibrio fibrisolvens and Megasphaera elsdenii (40 (link)), protozoa (41 (link)), and methanogenic archaea (42 (link)). The DNA standards Real-time PCR amplification and detection were determined using a Chromo 4™ system (Bio-Rad, CA, USA). The data of microbial population were transferred to log10 prior to statistical analysis.

Synthesized cDNA was used in real-time RT-PCR (Bio-Rad Chromo 4 system) experiments using iQ SYBR Green Supermix and analyzed with Opticon Monitor software according to the manufacturer’s instructions.
Real-time PCR was performed with an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, USA) using Power SYBR® Green PCR master mix (Applied Biosystems, USA) according to the protocols provided by the manufacturer. Briefly, PCR was performed in a final volume of 20 μl including 10 ng sample cDNA, 5 μM specific forward and reverse primers, and 10 μL Power SYBR® Green PCR master mix. PCR reactions consisted of an initial denaturating cycle at 95°C for 10 min, followed by 40 amplification cycles: 15 s at 95°C and 1 min at 60°C. The primers were used as Tables 1 and 2. Results were presented as levels of expression relative to those of controls after normalization to GADPH using the 2^-ΔΔCT method. Analysis was carried out in triplicates.

Quantitative real-time PCR analyses were performed using a CHROMO 4 System (Bio-Rad). The reaction mixture comprised Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, France), 0.5 μmol L⁻¹ of each primer, and the respective standardized cDNA as a template. Target gene copy numbers were normalized against the housekeeping genes hypoxanthine phosphoribosyltransferase (HPRT) and β2 microglobulin (B2m). Cytokine and chemokine genes evaluated were il1b, il6, il10, il12p70, il17a, il23, ifng, tnfa, tgfb, cxcl1, baff, april, gmcsf; the transcription factors studied were foxp3 and rorgt; epithelial barrier and IgA related genes were zo-1, occludin, and pIgR; mucin genes were muc1, muc2, muc3, muc4, muc6, and muc13. Primer sequences and PCR conditions are available upon request (E-mail: maria.urdaci@agro-bordeaux.fr). A negative control reaction without template was included for each primer combination.

Quantitative RT-PCR (qPCR) assays were run on a Chromo4 system (BIO-RAD) using GoTaq SYBR Green PCR kit (Promega, Madison, WI). Each reaction was performed in a final volume of 25 μl containing one μl cDNA and 2 x SYBR Green PCR Master mix. The amplification profile was denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 10 sec, 58°C for 30 sec. At the end of the PCR cycles, melting curve analysis of the PCR products was performed to validate the amplification of the specific products. Primer pair of 4a-F and Ex6-R for Ex4a(+)WT1 amplification and Ex6-F and Ex7-R primer pair for total WT1 amplification were used, respectively. The expression levels of WT1 mRNA were calculated using the comparative Ct method (2^-ΔΔCt) with Actin as the reference gene. Primer sequences for qPCR are as follows: 4a-F, 5’-ATTCCATTGCCTTTC
CACAG-3’; Ex6-R, 5’-GACACCGTGCGTGTGTATTC-3’; Ex6-F, 5’-GATAACCAC
ACAACGCCCATC-3’; Ex7-R, 5’-CACACGTCGCACATCCTGAAT-3’; Actin-F, 5’-CCCAGCACAATGAAGATCAAGATCAT-3’; Actin-R, 5’-ATCTGCTGGAAGGT
GGACAGCGA-3’. Bcl-xL-F, 5’-GATCCCCATGGCAGCAGTAAAGCAAG-3’; Bcl-xL-R, 5’-CCCCATCCCGGAAGAGTTCATTCACT-3’; Bcl-2-F, 5’-CGCCCTGT
GGATGACTGAG-3’; Bcl-2-R, 5’-AGCCAGGAGAAATCAAACAGAGG-3’. MRP4-F, 5’-AGGACACTTGCCATTGGATTA-3’; MRP4-R, 5’-ACCCTTGCAACT
CCTCTCCAAG-3’.