Tree and plant root samples for RNA-Seq

Roots contain polysaccharides, polyphenols, soil inhibitors and active RNases, so careful sampling and fast preservation are critical.

1. Plan the experiment

• Choose the root type for your question: fine roots (under 2 mm) for nutrient uptake and stress; lateral roots for branching; tap roots for growth; root tips (1–2 cm) for cell division and gene expression.
• At least 3 biological replicates (independent trees or plants) per treatment, at the same developmental stage and the same time of day.
• Avoid decayed, dark brown or black, or damaged roots.

2. Prepare before going to the field

• Liquid nitrogen and cryovials (or RNAlater and RNase-free 2 mL tubes), RNase-free forceps and scissors, gloves, cool box with ice packs, 70% ethanol and an RNase decontaminant (for example RNaseZAP), labels and waterproof marker.
• Label tubes in advance: sample ID, species, location, root type, treatment, date and time, replicate.
• Clean tools between replicates; new gloves for each sample.

3. Dig and collect

• Remove soil gently with sterile brushes or forceps; do not pull or crush roots.
• Select healthy roots and cut 2–5 cm segments, or 1–2 cm white, actively growing tips.
• Collect 100–300 mg fresh root tissue per sample (more for low-yield species).

4. Remove soil quickly

Shake off loose soil, rinse briefly in RNase-free water if needed and blot dry. Do not wash for long, as this changes stress-gene expression. Aim for less than 2–3 minutes from digging to preservation.

5. Preserve immediately

Option 1: Liquid nitrogen (best)

Put roots straight into cryovials and snap-freeze. Keep in liquid nitrogen or on dry ice during fieldwork, then store at −80 °C.

Option 2: RNAlater (when liquid nitrogen is not available)

1. Cut roots into 0.5–1 cm pieces and put them into RNAlater within about 1 minute.
2. Use 5–10 volumes of RNAlater: about 1 mL per 100 mg tissue. Every piece must be fully covered.
3. Close tightly and invert a few times.
4. Keep at 4 °C for 12–24 hours so RNAlater soaks in, then move to −80 °C.

6. Transport

Ship in an insulated box with ice packs (RNAlater samples) or dry ice (frozen samples). Keep below 30 °C and out of the sun. Avoid freeze–thaw.

7. Root-specific challenges

8. Quality targets for extracted RNA

Before extraction, pour off excess RNAlater and blot the tissue; carried-over RNAlater can interfere with some kits.