siRNA / miRNA gene silencing Human Caki-2

Though DNA quantification is but one small step in the multifaceted DNA sample preparation workflow, it can have large implications on the performance and validity of conclusions drawn from downstream assays. Major challenges include accuracy, precision, reproducibility, and detection of present contamination. Among UV spectrophotometry, fluorescence and real-time PCR based methods, the quantification method should be chosen based on the requirement of the downstream assay.

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DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

RNA-Seq is a method to sequence RNA by applying Next Generation Sequencing (NGS). The quality of RNA is critical for the success of RNA-Seq. The integrity of RNA is measured by the RNA integrity number (RIN). RIN is computed from RNA electrophoresis and electropherogram profiles (the peak area of the 28S rRNA should be approximately twice the peak area of the 18S rRNA). If you get the RIN value lower than 7, the possibility of getting the low quality of RNA-seq data is high. To get a high quality RNA, it is better to work with fresh samples or snap-freeze the tissues in liquid nitrogen as quickly as possible and store them at -80°C until further use. Make sure designated areas and all your filter tips, microfuge tubes, plastic, and glassware are RNase-free.

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