Nucleic Acid Analysis

A New Edge in Bisulfite Conversion

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Aberrant methylation events have significant impacts in terms of incidence of cancer and development disregulation. Researchers studying DNA methylation are often working with DNA from “difficult” tissues such as formalin-fixed, paraffin embedded tissues, which characteristically yield DNA that is more fragmented than that purified from fresh tissue. Traditional methods for bisulfite conversion involve a long protocol, harsh chemicals, and generally yield highly fragmented DNA. The DNA fragmentation may significantly impact the utility of the converted DNA in downstream applications such as bisulfite-specific PCR or bisulfite sequencing.

An ideal bisulfite conversion system enables complete conversion of a DNA sample in a short period of time, provides high yield of DNA, minimally fragments the DNA, works on a wide range of input DNA amounts (from a wide variety of sample types), and, while we’re at it, is easy to use and to store. Whew! That’s quite the list.

Continue reading “A New Edge in Bisulfite Conversion”

DNA Purification, Quantitation and Analysis Explained

Posted on by Isobel Maciver

WebinarsYesterday I listened in on the Webinar “Getting the Most Out of Your DNA Analysis from Purification to Downstream Assays”, presented by Eric Vincent–a Product Manager in the Promega Genomics group.

This is the webinar for you if you have ever wondered about the relative advantages and disadvantages of the many methods available for DNA purification, quantitation and analysis, or if you are comparing options for low- to high-throughput DNA purification. Eric presents a clear analyses of each of the steps in a basic DNA workflow: Purification, Quantitation, Quality Determination, and Downstream Analysis, providing key considerations and detailing the potential limitations of the methods commonly used at each step.

The DNA purification method chosen has an affect on the quality and integrity of the DNA isolated, and can therefore affect performance in downstream assays. Accuracy of quantitation also affects success, and the various downstream assays themselves (such as end-point PCR, qPCR, and sequencing) each have different sensitivities to factors such as DNA yield, quality, and integrity, and the presence of inhibitors. Continue reading “DNA Purification, Quantitation and Analysis Explained”

Working with RNA

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Set up a lab RNA Zone

Working with RNA can be a tricky thing…it falls apart easily, and RNases (enzymes that degrade RNA) are ubiquitous. Successfully isolating RNA and maintaining its integrity is critical, especially when sensitive downstream applications are used (e.g., RNA-Seq).

Good techniques for RNA handling are simple to employ but crucial for success. All RNA purification and handling should take place in an RNase-free, RNA-only zone of the lab. Segregating RNA work from protein and DNA purification and handling will help minimize the potential for RNase contamination and help keep your RNA intact. Only buffer and water stocks treated to be RNase-free should be kept in the RNA area of the lab, and gloves should be worn at all times to prevent accidental contamination. Tools and equipment such as pipets, tips, and centrifuges should be designated for use only in the RNA zone as well. The location of the RNA zone in the lab is also important. Keeping traffic to a minimum and moving the RNA zone away from doors, windows, and vents can also help minimize contamination.

Using an RNase inhibitor can also help safeguard your samples from RNase degradation. These inhibitors can bind to any RNases that may have been introduced into your sample and prevent them from cutting the RNA present.

Water and buffer stocks can be a source of RNase contamination. Several stocks from an RNase-free zone in an academic lab showed RNase activity. Recombinant RNasin® inhibitor protected all RNA samples from degradation.

Fixed in the Past, Focus on the Future

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“I would do more with my samples, but it’s just not possible…I know there’s probably a wealth of information in there, but there is just no way to get it out…I’ve got blocks of tissue sitting in the lab, experiments I want to run, but no good way to get clean nucleic acids out.”

These are a few of the comments I heard when talking with scientists at the American Society of Human Genetics meeting last week in Montreal. They, and countless other researchers, are sitting on a treasure trove of information that may have been locked away a few months ago, a few years ago, or decades ago. I’m referring to formalin-fixed, paraffin-embedded (FFPE) tissue blocks. It is estimated that there are upwards of 400 million tissue blocks archived globally and scientists are clamoring to find ways to best utilize nucleic acids derived from these tissues in applications like qPCR, microarrays, and next generation sequencing.1  Continue reading “Fixed in the Past, Focus on the Future”