Quantitative extraction of RNA from tissues high in secondary metabolites

Protocol

 

Author

Virginia Matzek

Overview

This protocol outlines how to quantitatively extract RNA from plant tissues high in polyphenolic compounds and polysaccharides.

Background

This technique is useful when the experimenter wishes to know how much RNA is contained in a plant tissue sample, e.g., to know how much nitrogen or phosphorus is contained in an active nucleic acid pool. The protocol has several steps: extraction of finely ground frozen plant material in a buffer; precipitation of nucleic acids from the supernatant on to silica particles; elution of nucleic acids; removal of DNA; quantitation of RNA with Ribogreen on a fluorimeter. Ribonuclease-free solutions and labware are used throughout to protect RNA from degradation. This protocol was developed for plant tissues high in secondary metabolites, specifically pine needles.

Materials/Equipment

  • Vortex mixer
  • Balance
  • Microcentrifuge
  • Water bath (55∘C)
  • Fluorimeter
  • Snap-cap RNase-free microtubes (1.5 ml)
  • Dry ice
  • Mortar and pestle
  • Liquid nitrogen
  • Sodium chloride (NaCl)
  • Polyvinyl pyrrolidone (PVP)
  • Ethylenediaminetetraacetic acid (EDTA)
  • Sodium acetate
  • Sodium dodecyl sulfate (SDS)
  • Beta-mercaptoethanol ( -ME)
  • Aurintricarboxylic acid (ATA)
  • Sodium iodide (NaI)
  • Ethanol
  • Superase-In
  • Tris-EDTA (TE) buffer
  • DNase
  • RNA standard
  • Ribogreen
  • Diethylpyrocarbonate (DEPC)

Procedure

Plant material preparation

1. To avoid degradation of plant RNA by endogenous ribonucleases, live plant tissue must be instantly frozen in liquid nitrogen at the time of harvest, remain frozen through grinding, and thaw only in the extraction buffer.

2. Choose healthy plant material unaffected by diseases or herbivory. Plunge tissue sample into liquid nitrogen immediately after harvest. Store at a maximum temperature of -20∘C.

3. To keep tissue frozen while grinding, use a ceramic mortar and pestle thoroughly cleaned with 95% ethanol. Embed mortar in a bowl of crushed dry ice and keep handy an open dewar of liquid nitrogen. Pre-chill pestle and a metal spatula in dry ice until ready to grind plant material. Label a receptacle (e.g., microtube) for the ground tissue, and pre-chill it in dry ice or liquid nitrogen. When everything that will touch plant tissue is at dry ice temperature or lower, pour frozen plant material into mortar and grind until a fine powder. Avoid breathing on plant tissue, which adds ice to the sample. Use spatula to scrape material into test tube and replace in -20∘C freezer. If material stays frozen, it will be easy to clean the mortar; only melted tissue sticks to mortar. If material thaws during grinding, discard it and clean mortar with ethanol.

4. This protocol yields a measurement of RNA content per unit fresh weight. If a dry-weight basis is desirable, an aliquot of the frozen tissue should be separated and weighed fresh before drying at 55∘C to a constant weight, to determine a fresh-weight/dry-weight conversion.

Preparation of buffer and silica slurry

1. Prepare an extraction buffer consisting of:

2.0 M NaCl

2 % PVP

25 mM EDTA

100 mM sodium acetate

3% SDS

2% beta-ME*

2mM ATA*

* added immediately before use

Check the pH of the buffer after adding the first four chemicals; if not pH 5.5, adjust with a drop of HCl. Then add SDS. This can be stored indefinitely, but the beta-ME and ATA must be added just prior to using the buffer.

2. Prepare a 50% (v/v) silica slurry. First wash the silica in RNase-free water, by shaking 10 g silicon dioxide in 100 ml RNase-free water. Let the solution settle 30 minutes, transfer the supernatant to a new flask, and let the supernatant settle another 30 minutes. Then transfer the supernatant to a large centrifuge tube and pellet the silica fines that remain suspended. Add a volume of RNase-free water equal to the estimated volume of the silica pellet to complete the slurry.

Extraction and quantitation of RNA

1. In a fume hood, put 300 l extraction buffer in 1.5 ml tube, and tare before adding ~25 mg sample. Add frozen tissue sample, vortex vigorously and reweigh to record exact sample mass. Flash freeze in dry ice until all tubes are weighed and ready.

2. Transfer samples to hot 55∘C bath for 10 minutes. Spin 10 min and transfer supernatant to a new tube.

3. Re-extract tissue pellet with an additional 50 l extraction buffer, repeating the warm bath, spin, and supernatant transfer steps in #3. The second extraction is critical for quantitative recovery of RNA; yields improve ~15-20% with the second extraction.

4. Precipitate nucleic acids with 350 l 6M NaI and 875 l ethanol. Invert to clear tube.

5. Add 25 l silica slurry to tube. Vortex thoroughly, rest tube while other samples are being vortexed, then vortex again thoroughly. Spin 2 minutes.

6. Pour off supernatant and wash silica pellet twice with 70% ethanol, resuspending pellet thoroughly each wash and repelleting with 2-minute spins. After 2nd wash, invert tube on paper towel to remove residual ethanol.

7. When tube and pellet are dry, add 10 l Superase-In (1U/ l) and 1.59 ml TE buffer. Elute nucleic acids in 55∘C bath, removing tube for occasional vortexing. Spin 2 minutes.

8. Prepare an RNase-free 96-well plate with 20 l DNase in each well. Make a standard curve (0,1,2,4,5,10,15,20 l of RNA standard in TE buffer), using TE buffer to equalize well volumes to 20 l. Transfer 20 l of each sample in 3 replicates to remaining wells.

9. Incubate well plate 30 minutes, then add Ribogreen (high-range) and incubate 30 minutes in dark. Run on fluorimeter at Ribogreen standard excitation frequencies.

Notes and troubleshooting tips

In general, solutions should be prepared in RNase-free glassware, using RNase-water (which can be purchased ready-made or prepared in the lab with diethylpyrocarbonate). However, the sample RNA is protected from RNases by the components of the extraction buffer, so the critical phase to avoid destruction of RNA is after precipitation of nucleic acids (step 6).

Grinding is the most important step in extraction when quantitative recovery is desired. The finer the powder, the more complete cell lysis will be possible.

Tissue should stay green through the extraction process. If tissue is browning, it means oxidation of polyphenolics is occurring; buffer may require increased concentration of PVP, beta-ME, or ATA (reducing agents).

DNA also binds to the silica, so Superase-In is required in step 7 to remove it. However, only about 15% of the DNA in the tissue binds to the silica in this method, so it is not a good method for quantitative recovery of DNA.

For quality control, it is recommended that the experimenter prepare a large quantity of frozen ground tissue of the same plant type as the samples for use as a “standard.” (Because it will not be possible to know how much RNA is in this tissue, it is not really a standard, but it is a useful background against which to test the relative recovery of pure RNA.) Spike tissue samples with pure RNA and test against pure RNA recovery to see how the presence of polysaccharides and phenolics affects RNA recovery.

Literature references

Matzek V, Vitousek PM (2009) N:P stoichiometry and protein:RNA ratios in vascular plants: an evaluation of the growth-rate hypothesis. Ecology Letters 12, 765-771.

Ainsworth C (1994) Isolation of RNA from floral tissue of Rumex acetosa (Sorrel). Plant Molecular Biology Reporter 12, 198-203.

Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter 11, 113-116.

Dong JZ, Dunstan DI (1996) A reliable method for extraction of RNA from various conifer tissues. Plant Cell Reports 15, 516-521.

Kolosova NB, Miller S, Ralph BE, Ellis CD, Ritland K, Bohlmann J (2004) Isolation of high-quality RNA from gymnosperm and angiosperm trees. Biotechniques 36, 821-824.

Lonneborg A, Jensen M (2000) Reliable and reproducible method to extract high-quality RNA from plant tissues rich in secondary metabolites. Biotechniques 29(4), 714-718.

Wang SX, Hunter W, Plant A (2000) Isolation and purification of functional total RNA from woody branches and needles of Sitka and white spruce. Biotechniques 28, 292-296.

Health, safety & hazardous waste disposal considerations

Beta-mercaptoethanol and diethylpyrocarbonate are hazardous substances requiring extreme caution. Preparation of DEPC-water, preparation of the extraction buffer and extraction of the sample (steps 1-6) should be performed in a fume hood, and care should be taken with contaminated gloves, glassware, and laboratory equipment.

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