When isolating RNA, good lab technique is essential. RNA is more labile in comparison to fixed and sacrosanct DNA. RNA, with a ribose sugar backbone, contains highly reactive hydroxyl (C-OH) groups, ensuring that strands are continually made, broken down and reused. Isolated RNA quickly degrades and is easily contaminated by resistant enzymatic ribonucleases (RNases), which are found everywhere.
In this edition of Bench Tips, we spoke with Dr. Jack Dumbacher, chairman and curator in the Department of Ornithology and Mammalogy at the California Academy of Sciences in San Francisco, about his lab’s current work isolating transcriptome and small RNA for the identification of metagenomic viral sequences. Contrary to the typical view of viruses as pathogens, viruses may also have mutualistic or symbiotic relationships with their hosts. Understanding these evolutionary relationships in birds is one of Dumbacher’s research goals.
“Typically with virus particles you’re after low-abundance RNA,” says Dumbacher. “Ribosomal RNA (rRNA) can be a huge portion of what you initially pick up. For good recovery, we try to increase our RNA yield by taking big samples in the field and preserving them as carefully as possible.”
Optimize RNA preservation
The Academy’s collecting expeditions often take place in remote areas renowned for their biodiversity, such as tropical forests or coral reefs. A big challenge for Dumbacher’s team is the inability to snap-freeze samples with liquid nitrogen because of the lack of refrigeration. (RNA steadily degrades in ethanol, a common preservative for DNA samples.) Instead samples—swabbed from the bird’s cloaca, the rear digestive opening—are placed in RNA stabilization reagents or other mixtures containing guanidinium thiocyanate (GITC) buffer at a 4 M concentration. RNA isolation in liquid samples can be difficult using some RNA stabilization reagents because of the salt content. When maintaining a chain of refrigeration is not an issue, the best way to preserve RNA is to immediately freeze samples after collection with liquid nitrogen and store samples at -80°C.
Ensure good separation
Traditionally, GITC buffers have been used to lyse cells and viral particles in RNA extractions and simultaneously prevent RNase enzymatic activity by denaturing RNA temporarily. In such cases, some intact viral separation—like filtration—must be performed before lysing to avoid a soup of nucleic acids. The samples collected by the Dumbacher team are predominantly in liquid form—little homogenization is required, but good separation is important. In the field, the team uses 0.2 µ filters—similar to those found in spin columns—to filter through smaller virus particles. Larger particles, like intact cells, are left behind. Other labs work with frozen tissue samples that must be homogenized quickly and thoroughly for good recovery. Regardless of the chosen method, some amount of genomic (gDNA) will always remain. Just how much gets through depends largely upon a user’s experience and technique.
Minimize RNase exposure
In the lab, eliminating contact between endo- and exogenous RNase and purified RNA is always a concern. Early in the RNA purification process, external sources of RNase are less of a threat. But after RNA preparations are no longer protected by denaturants like GITC solutions, and RNA is purified, you want to avoid activating RNases. Protein RNase inhibitors sequester remaining RNases and are suitable for most applications, especially those containing endogenous RNase. As a precaution, whenever equipment is touched—glassware or pipettes—gloves should be changed. (The recommended incubation time for glassware and utensils used in RNA purification is at least four hours at 180°C to 200°C.) The Academy’s Comparative Genomics Lab uses DEPC-treated water instead of molecular-grade water, when possible. DEPC water inactivates RNase through histidine modification of bases. When water is made in-house, autoclaving DEPC water beforehand, to degrade DEPC, is recommended.
Increase RNA yields
If you don’t already spec your extractions, start keeping track. RNA yields vary widely from different tissues and samples. For Dumbacher, every sample collected is typically low in RNA. The amount of RNA obtained depends on when a bird last ate, what it ate, and whether other chemicals or bacteria were at play, for instance. “Even when we use samples that have very low RNA, it’s pretty impressive when you build your libraries, and you see how many sequences you obtain on the other end,” says Dumbacher. (His group is currently using next-generation sequencing technology to isolate viral sequences.)
To increase RNA yields in (previously RNA-robust) tissue samples, avoid excessive homogenization or heat. Homogenizing in bursts of 30 seconds with 30-second rest intervals can improve RNA recovery. Also, eluting with more water releases more RNA from the membrane when using silica spin filters. Regardless of the downstream technology used in your lab, successful analysis depends on good RNA isolation technique. Practice makes perfect. Happy extracting!
The image at the top of the page is from Bio-Rad's Experion mRNA Assay.