The isolation of high-quality DNA and RNA samples from plants can be challenging due to the presence of inhibiting and damaging phytochemicals. Thus, it is not possible to recommend a single protocol that works for all samples. In any case the RNA samples should be DNAse treated, and QC-ed on a Bioanalyzer for sample integrity and via Nanodrop for purity. Please see sample requirements.
For many species and many types of samples the Qiagen RNeasy Plant MiniKits (cat. no. 74903) have been applied successfully. For RNA-seq and Tag-Seq projects this kit has to be used in conjunction with the Qiagen RNase-Free DNase Set (cat. no. 79254) . We recommend to isolate the RNA, then perform the DNAse digestion on the isolated RNA, and then clean up this reaction once more with the RNeasy kit. Alternatively, you could use the Zymo RNA clean & concentrator 5 kit with DNAse (cat. no. R1013) for the DNAse digestion and clean up.
The NEB Monarch Total RNA Miniprep Kit comes with an “RNA Protection Reagent” that can be added during the mechanical disruption of plant samples.
- Always perform at least two spin column washes (with the kit wash buffer) after binding of the lysed sample to the column matrix. Perform the “optional” steps described in the kit manual. Also, add a short “dry spin” of the column after the washes and before the elution buffer addition to avoid carryover of the ethanol wash buffer.
- Avoid glycogen (often used as a co-precipitant).
- To achieve the cleanest RNA isolations only use at most half the sample amount of the maximum recommended by the manufacturer.
Avoiding Batch-Effects:
Both sample storage conditions and details of the RNA-isolation protocols are well-known to introduce technical variations into RNA-seq data. Because of this, it is recommended to:
- Isolate the RNA-samples in one batch.
- If RNA-isolations need to be carried out in several batches, they should be carried out by the same person using the same batch of reagents
- If RNA-isolations need to be carried out in several batches, the samples should be randomized between the RNA isolation batches (worth discussing with a statistician or the Bioinformatics Core).
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