If you’ve ever loaded a sample on a gel and ended up with a smear, or used capillary electrophoresis to check your RNA and saw something that looked like a seismograph tracing instead of distinct peaks and valleys, you understand the importance of sample preparation. Although at first glance analyte purification appears to be a routine task, low sample quality may lead to uninterpretable results, wasting time and resources. Early consideration of sample type, experimental scale and downstream applications are essential to avoid costly mistakes. This is especially important when the sample is small and precious, as is often the case with human samples.
Do-it-yourself nucleic acid isolation techniques typically rely upon organic solvents such as phenol. Alternatively, commercial kits include all necessary reagents and use a solid medium for selective binding of DNA or RNA. Two purification options are common in reagent kits: silica and anion exchange media. Both silica and anion-exchange columns are convenient and adaptable to automation using liquid handling robotics. Reagent kits may be designed for generic sample types, or specialized according to the source of material (such as yeast, bacteria, viruses, or blood and tissue).
Modern research often relies upon genomic and proteomic techniques, coupled with computational analysis, to obtain an integrated network of information about complex biological systems. Simultaneous isolation of DNA, RNA and protein is often required for these studies, which may span in vitro, in vivo and clinical data. The holy grail for these studies is a single reagent or extraction system that simultaneously isolates DNA, RNA, and protein without compromising yield or sample quality. Historically, guanidinium thiocyanate-phenol-chloroform extraction has been the go‑to solution. However, if you’ve ever tried to obtain a useful protein sample from this technique, you know that the reality doesn’t always live up to expectations. Protocol modifications have been introduced to improve protein recovery, with some reported success, although for many this achievement remains a biological urban legend. Newer commercial kits like Allprep from QIAGEN have proven more successful at the task, but yields of DNA and protein have been shown to be lower (1). When sample quantity is not limiting, however, triple-analyte kits are an excellent time-saving option.
Techniques that have been used for decades to isolate analytes may not always be the best solution for newer applications, such as next-generation sequencing, epigenetic studies and miRNA isolation. One case in particular highlights the pitfalls of using validated reagents for non-validated applications. The authors analyzed miRNA abundance in both adherent and trypsinized cells. They found that specific miRNA molecules were rapidly degraded, within 1 hour, after trypsinization, leading to the conclusion that these miRNA molecules played a role in the response to changes in cell adhesion (2). Upon further examination, the authors determined that their miRNA extraction reagent, when used with a small amount of starting material, led to the preferential loss of low GC-content molecules during sample preparation. The end result was an erroneous conclusion, a retracted journal article and helpful “heads-up” for anyone using the reagent for similar purposes (3). The incident provided important lessons: the old standby technique does not always perform as expected with new applications, and if your results depend upon the isolation of non-standard analytes, it might be best to use specialized, validated kits or reagents designed specifically for the molecule of interest.
- 1. Mathieson, W. and Thomas, G.A. (2013) Simultaneously extracting DNA, RNA, and protein using kits: is sample quantity or quality prejudiced? Anal. Biochem 433, 10.
- 2. Kim, Y.K. et al. (2011) Cell adhesion-dependent control of microRNA decay. Mol. Cell 43, 1005.
- 3. Kim, Y.K. et al. (2012) Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells. Mol. Cell 46, 893.