Cfx96 qpcr thermal cycler

Fresh retinas were dissected from the eyes of anesthetized P1, P5, P10, and adult B6/J and A/J mice and placed into RNAlater (BioRad), taking care to avoid RNase contamination. Adult samples were comprised of both retinas from individual animals, while developmental samples were comprised of pooled retinas from mice in the same litter (∼3–4 mice). RNA was purified from each sample using an RNeasy Mini Kit (#74104; Qiagen), and single stranded cDNA was produced from these samples using an iScript cDNA synthesis kit with oligo (dT) and random primers (#1708890; BioRad). Sso Advanced Universal SYBR Green Supermix was used to determine the relative expression of full-length Dixdc1 transcripts (using primers directed to the common 3′UTR) to the amount of the truncated transcripts via qPCR. Adult and developmental samples were assessed separately. For each gene, every sample was run on a single plate in triplicate using the CFX96 qPCR thermal cycler (BioRad). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and β-2 microglobulin (β2m) were used as internal control genes for normalizing Dixdc1 expression to compare quantities across samples (as described in Keeley et al., 2012 (link)). Details for the qPCR primers are listed in Table 3.

Total RNA was extracted using Direct-zol™ RNA MiniPrep kit (Euroclone) and the purified RNA was reverse-transcribed using iScript™ cDNA synthesis Kit (Biorad) according to the manufacturer’s instructions. Each qRT-PCR was performed in triplicate on a CFX96 qPCR Thermal cycler (Biorad) and the results were normalized to GAPDH and TBP mRNA levels. The specific primers sequences used are provided in Supplementary Table S3.

The quantification of specific bacterial groups (Firmicutes, Bacteroidetes, Bacteroides, P. dorei, Bifidobacterium sp., Clostridium cluster IV, Enterococcus sp., Lactobacillus sp. Proteobacteria and Prevotella sp.) was determined by quantitative real-time PCR (qPCR). The specific primers used are indicated in Table S1. Reactions were performed using SsoFastTM kit Eva Green Supermix (BioRad, Hercules, CA, USA), 0.5 μM of forward and reverse primer and 100 ng of DNA on a CFX96 qPCR thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). All PCR reactions were carried out in duplicate, and the PCR conditions are shown in Table S2. Reactions containing all the components except template DNA were included as negative controls. The denaturation step occurred at 95 °C, for 10 min. The number of cycles was identical for all target groups (45 cycles). The melting curve was performed by increasing the temperature 0.5 °C each 2 s from 72 °C to 95 °C.
Standard curves were generated using plasmid DNA (pCR ™ 2.1-TOPO) with inserted 16SrRNA gene of each bacterium. The cloning process was achieved using competent Escherichia coli MACH1 (Invitrogen) following the instructions of the vector manufacturer (Invitrogen). Four replicates of the plasmid DNA dilutions in order to obtain the number of copies that ranged from 10¹⁰–10 were prepared. Data are expressed as Log₁₀ DNA copies/g feces.