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Best practices for RNA-seq success, part I: Optimizing sample prep
To all NGS newbies and sequencing savants alike...welcome! In this article, you'll learn tips and tricks on how to achieve robust and reliable RNA-seq data, starting with RNA sample prep. How do I prevent my RNA from degrading after I collect it? What is the best purification kit for my research application? When is it necessary to perform rRNA depletion prior to cDNA synthesis? All these questions and more will be answered in our blog! Written in collaboration with our Technical Support Scientists, Drs. Marianne Rivkin and Bakhyt Zhumabayeva. Special thanks to them and Dr. Peiyong Huang, manager of the Technical Support team.
Research is so unpredictable. There are periods when nothing works and all your experiments are a disaster and all your hypotheses are wrong."
—Francis Collins, former director of the NHGRI and project head of the Human Genome Project
Let’s face it. Research really is unpredictable. A scientist may spend hours planning experiments and adopting different analysis strategies depending on the organism being studied and their research goals…only to start all over again if even one thing goes wrong. RNA-seq is certainly no exception.
There is no optimal, “one-size-fits-all” experimental template for the variety of different applications and analysis scenarios in which RNA-seq can be used. Every RNA-seq experiment could potentially have different optimization methods for transcript quantification, normalization, and ultimately differential expression analysis. However, all RNA-seq workflows can essentially be boiled down to 3 steps: RNA extraction and sample preparation, cDNA synthesis and library preparation, and finally, data analysis.
While each step of the experimental setup is of critical importance to get the most reliable results possible, sample prep stands at a pivotal point in experimental success.
While RNA extraction is a notoriously daunting and oftentimes frustrating task, it is not impossible. As RNA can be unstable and highly susceptible to degradation, the number one priority is to get the RNA out and stabilized as quickly as possible (ideally at the time of collection). Once that has happened, downstream storage and processing are much more manageable. Common methods for sample stabilization include snap freezing with liquid nitrogen, dry-ice ethanol baths, immediate storage in a -80°C freezer, or submerging the sample in a stabilization reagent meant to inactivate nucleases. However, these approaches have drawbacks, such as freeze-thaw damage of nucleic acids or inhibiting/interfering with cDNA synthesis. We instead recommend immediate solubilization in a lysis buffer that contains RNase inhibitors (i.e. the RNA Lysis Buffer included in our purification kits).
The wide variety of RNA purification methods available can make it difficult to decide which one to use. Two very important points to keep in mind while making your selection are...
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