Can anyone suggest me a better protocol for extraction of total RNA from Arabidopsis dry seeds? I tried with commercially available kit for extraction but the yield was very less. I used around 200mg of tissue and crushed in liquid Nitrogen and followed the protocol. How to avoid carbohydrate and lipid contamination?
pL. SEE THE REFERENCE
Phytochem Anal. 2009 Mar-Apr;20(2):139-42. doi: 10.1002/pca.1108.
An optimised, small-scale preparation of high-quality RNA from dry seeds of Davidia involucrata.
Qi G1, Li JT, Ruan QP, Yang J, Su ZX.
Author information
1Sichuan Provincial key Laboratory of Environmental Science and Biological Diversity Conservation, China West Normal University, Nanchong, Sichuan, People's Republic of China.
Abstract
INTRODUCTION: The dormancy of Davidia involucrata seeds normally lasts for an extended period of time and because of this unique property the species is an excellent model for studying the molecular mechanisms of plant dormancy. The use of minimal plant material is desirable for RNA extraction since D. involucrata is a rare plant and it is relatively difficult to collect large amounts of seeds in order to perform molecular biology studies.
OBJECTIVE: To improve the quality of RNA obtained from seeds of D. involucrata by eliminating the oxidation of polyphenols during extraction and by preventing polysaccharides and other impurities from being extracted.
METHODS: A previously described method was modified by the addition of 4% (w/v) poly(N-vinyl-2-pyrrolidone) to the dry seeds during grinding and by adding 5% (v/v) beta-mercaptoethanol and 28% (v/v) ethanol to the extraction buffer. Two further centrifugation steps (5000 and 8000 rpm) were utilised and one-seventh volume of ethanol was incubated with the supernatant at 4 degrees C for 2-3 h prior to the precipitation of RNA.
RESULTS: Following these modifications, an effective method was established for total RNA extraction from a small amount of dry seeds of D. involucrata. The isolated RNA was shown to have high purity and integrity by gel electrophoresis and spectrophotometry, and was confirmed to be suitable for RT-PCR and the construction of cDNA libraries.
CONCLUSION: The modified method reduced the amount of seeds required for extraction of total DNA and was beneficial for preserving the endangered species.
Copyright (c) 2009 John Wiley & Sons, Ltd.
PMID: 19140111 [PubMed - indexed for MEDLINE]
Please consult the attached protocol published in Biotechnique. Please note that, most of the RNA isolation kits fail to yield desirable quality+quantity of RNA required for downstream analysis. A manual LiCl-Phenol method is more suitable for starch-rich seeds.
Hi, I've been using white quartz sand (Sigma-Aldrich) as a grinding helper (I use mortar and pestle), that improved notably the final yield from dry seed samples (Arabidopsis) even using commercial kits (I use a non-column one, MasterPure™ Complete DNA and RNA Purification Kit). With this kit I get a very low contamination from lipids and CH, but the best way to get rid of those is to re-precipitate with isopropanol (at least that's what worked for me). Good luck!
Please see the materials and method of the following paper:
Coevolution Pattern and Functional Conservation or Divergence of miR167s and their targets across Diverse Plant Species.
Barik S, Kumar A, Sarkar Das S, Yadav S, Gautam V, Singh A, Singh S, Sarkar AK.
Sci Rep. 2015 Oct 13;5:14611. doi: 10.1038/srep14611.
You can see good quality of RNA (Fig. 8) isolated from apple seeds (very rich in phenols); This works even better for Arabidopsis.
Best
You can refer the material and methods of the following paper, it may help:
Characterization of 5′UTR of rice ClpB-C/Hsp100 gene: evidence
of its involvement in post-transcriptional regulation
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