We need to study expression of ergosterol biosynthesis genes in Fusarium oxysporum
I would not use a kit but try it the old fashioned way.
For S. cerevisiae we use glass beads and Guanidium thiocyanate, followed by vortexing and a 10 minute incubation at 65ºC. This tends to give you much higher yields than any kit and you also get more smaller RNA species. I can send you the protocol if you wish
Hello Dr. Dananjaya,
Dr. Chomczynski has asked me to respond to the email question that you recently posted at Research Gate.
As you might expect, we have not had any direct experience working with Fusarium oxysporum; however, Molecular Research Center, Inc. has a number of reagents that could be used in this application. My first choice would be RNAzol-RT.
RNAzol-RT is a modification of the original single-step method for RNA isolation. I have attached an application note that discusses some of the differences between RNAzol-RT and TRI Reagent. The primary advantages of using RNAzol-RT are the lower levels of DNA contamination and the slight improvement in the recovery of total RNA.
TRI Reagent and RNAzol-RT utilize different chemistries. Proteins are partitioned into the organic fraction in TRI Reagent isolations by the initiation of the phase separation step following the addition of either chloroform or BCP. The RNA remains in the aqueous phase and it can be subsequently recovered by alcohol precipitation. In contrast, proteins and DNA are removed from the RNAzol-RT homogenate following the water precipitation step and the RNA again remains in the water supernatant and it also is recovered by alcohol precipitation.
RNAzol-RT provides some distinct advantages because it is possible to obtain total RNA or an enriched large RNA (> 200 bp) and small RNA (< 200bp) fractions. The large RNA protocol provides extremely clean RNA and this protocol is very useful when working with samples that contain large quantities of polysaccharide and proteoglycan (e.g. plant and bacterial samples). The large RNA protocol may be particularly useful with your fungal samples.
Since RNAzol-RT employs a water precipitation step, liquid samples as large as 0.4 ml may be added to 1 ml of RNAzol-RT. Since your samples will probably be harvested in some type of culture medium, RNAzol-RT would be ideally suited for these liquid samples.
Finally, although RNAzol-RT will provide high quality RNA, the most important step in the extraction process is providing a mechanism for the RNA to be accessible to the reagent. Therefore efficient sample homogenization and cell permeabolization is essential to good RNA recovery. I have included a second attachment to my email that provides some additional tips that may be helpful in your extractions.
I hope that you will find my comments useful in your studies. A detailed protocol for RNAzol-RT is available at our web site (www.mrcgene.com). Please contact me if you have any additional questions relating to this matter.
Best regards,
Bill Wilfinger, Ph.D.
Senior Scientist
Product Development and Technical Services
Molecular Research Center, Inc.
Norgen's Plant/Fungi RNA Purification kit
The attached paper will help in resolving your problem.
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