A Starchy Situation

Potatoes, bread, rice, pasta, and other starchy foods are some of the most comforting foods in the world, as they remind us of childhood meals and time with our families while providing us with an easy source of energy. These starch-rich plants are staples of the modern diet worldwide and there is great interest in the genetic manipulation of these crops. To study this, the growing field of food sciences is ever evolving and incorporating molecular techniques to study the genomes and transcriptomes of food crops to understand how to increase hardiness and generally boost yield. However, extraction of nucleic acids from starchy foods are notoriously difficult and optimized methods are required to eliminate inhibitors of downstream analysis methods, such as qPCR, RT-qPCR and Next-Gen sequencing.

complicated methods

Fortunately, scientists have come up with many approaches to separate nucleic acids and polysaccharides, but many of them are quite complicated. Historically, when working with starchy samples, researchers utilize buffers containing high salt and cetrimonium bromide (CTAB) to separate the nucleic acids from starch. CTAB is a surfactant that has the advantage of binding polysaccharides thus removing them from the solution and co-purifying with nucleic acids. Typically, CTAB-based procedures are then paired with further organic extraction methods, such as phenol: chloroform: isoamyl alcohol for DNA or TRIzol^® for RNA.

downstream extraction methods

Although effective, downstream extraction methods can be lengthy and result in sample loss if not carefully controlled and optimized. Fortunately, for RNA purification using TRIzol^®, the Direct-zol RNA Kits collection solves these issues. After CTAB treatment, samples can be extracted directly from TRIzol^® reagent with no phase separation. The resulting unbiased RNA is of high quality, and ready for downstream applications.

The following protocol has been successfully used by our customers.

Steps to isolate RNA from polysaccharide- and polyphenolic-rich samples:

1. Prepare Extraction Buffer: 2% CTAB, 2% PVP40, 25mM EDTA, 100mM Tris at pH 8, 2M NaCl and 0.5 g/L spermidine. Autoclave extraction buffer and then add 3% (v/v) β-mercaptoethanol (2- mercaptoethanol, BME) prior to extraction procedure below.
2. For each sample, pre-heat 1.2 mL Extraction Buffer in a 2 mL Eppendorf tube at 65°C in a water bath.
3. Homogenize 250 mg of sample in liquid nitrogen using a mortar and pestle and transfer powder to the pre-heated extraction buffer.
4. Vortex immediately for 1 minutes and place back in the water bath. Keep the sample in the water bath for at least 30 minutes and vortex once every five minutes.
5. Centrifuge at maximum speed (16000 x g) for 10 minutes and transfer supernatant to a new 2 mL microcentrifuge tube.
6. Add an equal volume of chloroform: isoamyl alcohol (24:1), then vortex for 30 seconds and centrifuge at maximum speed for 15 minutes at 4°C.
7. Pipette aqueous phase, without disruption of the white interphase, and transfer to a new 2 mL microcentrifuge tube.
8. Repeat the chloroform: isoamyl alcohol extraction (step 5 and 6).
9. Add an equal volume of ethanol (95-100%) to the aqueous phase, mix and load onto the Zymo-Spin column. Continue with the Direct-zol RNA Kit protocol (page 4, step 3) or the RNA Clean & Concentrator protocol (page 3, step 4). Perform all the steps at room temperature.

For RNA extracted by other protocols that are still contaminated with polysaccharides and other polyphenolics, the OneStep PCR Inhibitor Removal Kit can be used to remove inhibitors. This kit a simple, one-step procedure for removing PCR inhibitors such as polyphenolics, humic/fulvic acids, tannins, melanin, etc. As a substitute for a CTAB-based protocol, RNA extraction can combine the Direct-zol RNA kit followed by treatment with the OneStep PCR Inhibitor Removal kit.

While challenging, the above implementing the above steps will increase success for extraction of RNA for processing starchy foods. For any further clarification or support, feel free to contact the Zymo Research technical support team ( tech@zymoresearch.com).