Cell to ct kit

Cells were lysed and RNA was extracted from the lysate followed by RT performed using the Cell-to-Ct Kit (Life Technologies). A volume of2 μl of synthesized first strand cDNA was used for qPCR performed with a Universal SYBR green mix (BioRad) using a total reaction volume of 10 μl. RT-qPCR was performed using an Agilent HT7900 instrument (Applied Biosystems, Foster City, CA, USA). Primers were obtained from RealTimePrimers.com.
The qPCR steps consisted of 20 s at 95 °C and 40 cycles each for 3 s at 95 °C and 15 s at 58 °C and 15 s at 68 °C. Expression data were calculated using the comparative threshold cycle (C_t) method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. The C_t data for GAPDH was used to create ΔC_t values [ΔC_t = C_t (target gene)−C_t (GAPDH)]. Relative quantification values were calculated using the equation: 2^−ΔCt.

Relative gene expression was performed through mRNA isolation from MDA-231, MDA-468, and HEK293T cell lines using Invitrogen Cell to C_t kit (Life Technolgoies #4399002). Following the manufacturers’ recommendations, the cells were plated in a 96-well plate, and the untreated cells were grown to 75% confluency. For transfection, 0.1 μg of plasmid DNA was added with Fugene 6 transfection reagent in a 1:6 ratio (plasmid DNA to Fugene). Cells were then allowed 48h to recover before being lysed for mRNA isolation using an Invitrogen Cell to C_t kit (Life Technologies #4399002). The cells were then lysed, and RT-PCR was performed to produce cDNA using the reagents from the kit. After cDNA synthesis, qPCR was performed using TaqMan Gene expression primers (Table 3) and the TaqMan master mix provided with the kit (Applied Biosystems Foster City, CA, USA #4369016). The assay was performed in technical triplicate over three biological replicates. Results represent the average of the three biological replicates ± standard error of the mean (SEM).

Isolation of mRNA using an Invitrogen Cell to C_t kit (Life Technologies #4399002) was used for the relative gene expression in HEK293T and U2OS cells, following the manufacturers’ recommendations as previously described [3 (link)]. Cells were plated in 96-well plates and grown to 70% confluency before treatment with high glucose media. Cells were then lysed, and RT-PCR was performed to produce cDNA. Following cDNA synthesis, qPCR was performed using the appropriate TaqMan gene expression primers (XRCC1 Hs00959834_m1 FAM and IL-6 Hs00174131_m1 FAM) and TaqMan master mix. Each gene expression experiment was performed in technical triplicates with three biological replicates. Quantifications are represented as the mean of the three biological replicates $\pm$ the standard error of the mean (SEM).

For qPCR experiments RGCs were grown in 48 well plates, 3 wells were pooled per experimental condition. RGCs were treated with Gpc4 for the time indicated in the text (1 hour, 4 hours, 12 hours or 18 hours). RGCs were then lysed and cDNA prepared using the Cell-to-Ct kit (Life Tech Cat. 4402954). Reverse transcription reaction contained the following steps: 1 cycle at 37°C for 60 minutes, 1 cycle at 95°C for 5 min. The cDNA was then used for the qPCR reaction. Rat Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control housekeeping gene to normalize the Ct values obtained for the candidate gene primers. The qPCR reaction was performed using the power SYBR green PCR master mix (Life Tech Cat. 4402954) and contained: one enzyme activation step at 95°C for 10 minutes, 40 PCR cycles at 95°C for 15 s and at 60°C for one minute. Data was analyzed using the SDS 2.4 software (Applied Biosystems).

Primers for qPCR are listed in Additional File 2, Table S10. All primers were evaluated by construction of five step 1:5 dilution standard curves. By stability evaluation using the Cotton EST database RefFinder tool [60 ] across all independent isogenic clones and the respective parental RKO and HCT116 cell lines, TBP and HPRT1 were selected as endogenous reference genes. The cDNA was synthesized from total RNA using the Maxima H minus First Strand cDNA synthesis kit (Thermo Scientific) using the supplied random primers. Technical triplicate 20 μl qPCR reaction with 1× Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) and 0.3 μM of each primer were run on the StepOne Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Each assay included no-template controls, and each RNA sample was assessed for gDNA contamination by cDNA synthesis reactions where reverse transcriptase was omitted. For assessment of lentiviral knockdown efficiencies for TSPAN1 and PRSS23, all cDNAs were prepared directly from cell lysates using the Cell-to-Ct kit (Life Technologies), and specific TaqMan assays (ThermoFisher Scientific; Additional File 2, Table S7) were used to measure gene expression levels.