I am preparing an experiment in which I would like to sample roots of sunflowers grown in pots (substrate is soil) in later stages of the plant development. Can anyone tell me, what is the best way to quickly sample and wash roots so they could be of good quality for RNA extraction and subsequently for transcriptome analysis?
I also understand that it is not the same if one makes a mix of all roots, or choose a particular part of the root to sample, so any advice would be highly appreciated.
the exact protocol
**Sampling Roots:**
1. Carefully remove the sunflower plant from the pot, ensuring not to damage the roots.
2. Gently shake off the excess soil from the roots.
**Washing Roots:**
1. Rinse the roots under running tap water to remove the remaining soil particles.
2. For a more thorough cleaning, you can soak the roots in a bucket of water and gently agitate them. Repeat this process until the water remains clear.
**RNA Extraction:**
1. After washing, you should quickly move to the RNA extraction process to prevent RNA degradation⁵.
2. You can use a commercial RNA extraction kit, such as the miRNeasy Mini Kit⁵. This kit includes a protocol for isolating total RNA, including small RNAs, from plant tissues⁵.
3. The extraction process generally involves homogenizing the tissue, separating the phases, binding total RNA, washing, and finally eluting the RNA⁵.
**Choosing the Part of the Root to Sample:**
1. The part of the root you choose to sample can indeed affect the results of your transcriptome analysis¹.
2. It's important to consider the specific research question you're trying to answer. For example, if you're interested in how different parts of the root respond to certain conditions, you might want to sample different root sections separately¹.
3. If you're more interested in the overall root response, a mixed sample might be more appropriate¹.
Remember to always work in RNase-free conditions to prevent degradation of your RNA samples⁶. Also, it's crucial to validate your results with replicates and consider the developmental stage of the studied individuals².
(1) Isolation of total RNA, including small RNAs, from plant tissues. https://www.qiagen.com/us/resources/download.aspx?id=2bfdb422-5484-4186-98d4-e6d13fb0625f&lang=en.
(2) Transcriptome sequencing analysis of maize roots reveals the ... - Springer. https://link.springer.com/article/10.1007/s11104-021-04921-0.
(3) RNA Extraction: Principle, Procedure, Protocol and Importance. https://geneticeducation.co.in/rna-extraction-principle-procedure-protocol-and-importance/.
(4) A brief introduction to transcriptomics: from sampling to data analysis. https://omics.leeds.ac.uk/wp-content/uploads/sites/45/2021/03/A-brief-introduction-to-transcriptomics-from-sampling-to-data-analysis.pdf.
(5) A survey of best practices for RNA-seq data analysis. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0881-8.
(6) Transcriptome analysis of potato shoots, roots and stolons under .... https://www.nature.com/articles/s41598-020-58167-4.pdf.
(7) Total RNA Extraction: Easy Methods for Getting High-Purity RNA. https://bitesizebio.com/13529/total-rna-extraction-protocol/.
(8) A Simple and efficient protocol for isolation of RNA from different .... https://ndpublisher.in/admin/issues/IJAEBv9n1f.pdf.
(9) A Guide to RNA Extraction, RNA Purification and Isolation. https://rna.cd-genomics.com/resource/rna-extraction-guide.html.
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