Fragmented total RNA

[Yew]

Hi all,

Currently i have been facing this problem. My total RNA extracted from plant sample give high purity. But, the total RNA was fragmented. the 28s and 16s are clearly observed but with multiple bands below these two.

I wonder why this happen. I suspect the centrifugation force coz this. so i reduce the force from 15,000xg to 10,000xg. Still the same problem occur. During tissue lysis, i try to mix the extraction buffer with the pulverized sample just with gentle inverting for 1 min, is stead of vigorous shaking or vortexing. However, still no improvement.

Anyone have faced this b4? i really confused with this.

TQ.

[relaxin]

This is caused by RNase digestion. Unlike high Mol Wt genomic DNA, RNA is rarely fragmented by high centrifugal force or vortexing or homogenization using a Polytron. To avoid degradation, you may try to homogenize frozen tissue directly in extraction solution containing denaturing agent (guanidium isothiocyanate)to inactivate the RNase. If the tissue is too hard for homoenization directly, you can pulverize it in liquid nitrogen first.

[Yew]

Hi,

I really wonder if the RNase is so abundant in old tissue (my sample is ripe fruit). I tried increase 2x volume of extraction buffer (using same amount of tissue) for extraction, this problem is still persist. I have to mention that my colleague cn get a very intact RNA using the same protocol. Different hand may coz this?

Commercial kits are not working for my sample.

I am not considering guanidium buffer for extraction for the reason that high amount of plant polysacharides and polyphenolic compounds cnt be removed, although it is good for rnase inhibition. Usually, the strategy for removing these compounds is using high concentration of salt, like NaCl and KOAc (search around the Plant Molecular Biology Reporter, u cn find a lot of RNA extraction method from plant sample). With high concentration of salt, the activity of rnase is inactivated altogether. The following PCI and CI extraction will remove all proteins and isolated poly-compounds.

The whole process is truely logical. firstly lyse the tissue, then remove the poly-stuff, finally remove all other molecules by PCI. Thats y i suspect the fragmentation is due to mechanical force during the process.

[relaxin]

I am not familiar with plant RNA extraction. It seems that your protocol is working fine, at least for your colleague. It is true that different hands may have different result. It may be better if you can go through the protocol with your colleague and see what you have done wrong.

[Abhijeet Bakre]

I recall having seen a protocol using CTAB to remove the sugars seen by plant biologists atleast for DNA purification. I googled and found this for plants

A simple and efficient method for isolating RNA from pine trees
S Chang, J Puryear, J Cairney - Plant Molecular Biology Reporter, 1993 - Springer

Isolation of high quality RNA from bilberry ( Vaccinium myrtillus L.) fruit
Journal Molecular Biotechnology
Publisher Humana Press Inc.
ISSN 1073-6085 (Print) 1559-0305 (Online)
Issue Volume 19, Number 2 / October, 2001

I found that people actually have used CTAB across different plants and tissue types. You might want to google "CTAB for RNA extraction" and find something that works for your system.
Abhijeet