Fitamp general tissue section dna isolation kit

DNA methylation was analyzed using MS-PCR. Briefly, DNA was isolated from lungs (5 μg) using a FitAmp General Tissue Section DNA Isolation Kit (EpiGentek catalog no.: # Kit P-1003), after which the collected DNA was eluted in Tris-EDTA buffer in a total volume of 30 μl. The DNA was then quantified using a fluorescence quantification method, and 150 ng samples of the DNA were used to perform bisulfite conversion using the Methylamp DNA Bisulfite Conversion Kit (EpiGentek catalog no.: # P-1001). The conversion efficiency of bisulfite-treated DNA was determined with RT-PCR using two primer pairs and control DNA. The first primer pair was against bisulfite-converted DNA (β-actin), while the second was against unconverted DNA (GAPDH) in the same bisulfite-treated DNA sample. The DNA was shown to be >98% converted. qPCR was performed in duplicate using 1-μl aliquots of modified DNA and gene-specific primers designed rat genes. Data analysis was performed using qPCR Ct values to calculate the percentage MI for each target region: % MI = [1 – (2^{(Ct M-Ct uM)}/2^{(Ct M-Ct uM+1)})] × 100%. The MI measures the degree of methylation at a specific gene site; a higher MI indicates a higher degree of methylation. The MI for fully methylated DNA is defined as 100%. A relative comparison of the samples can be made based on the MI.

DNA from 2 to 6 mg tissue (brain, thymus, heart, lung, liver, spleen, adrenal gland, kidney, pancreas, muscle) was isolated with the FitAmp General Tissue section DNA Isolation Kit (Epigentek, Farmingdale, NY, USA) and 500–1000 ng DNA subsequently digested into deoxynucleosides with the EpiQuik One-Step DNA Hydrolysis Kit (Epigentek) according to manufacturer’s instructions.

A commercial tissue section DNA isolation kit (FitAmp General Tissue Section DNA Isolation Kit, catalogue no. P-1003, Epigentek Group Inc., Farmingdale, NY, USA) was used to isolate small intestinal DNA from adult offspring, according to the manufacturer’s instructions. Sample lysis was performed on a dry bath, set at 65 °C for 90 min. The genomic DNA was then captured and purified using spin columns prior to washing out with an elution buffer.