I have some of the protocols on brown algae but the yield is always less in my experiments. I am a bit worried that this is only the case for me, or is the yield from brown algae generally less?
I want to know if anyone has any experience with this and can they share with me how much should I use to get sufficient RNA for cDNA and real time qPCR?
I have not worked on brown algae but have on rhodophytes besides microalgae my research focus. In general, extracting RNA from algae is challenging, particularly so for some lineages. My lab has developed a protocol that works for us most of the time. The protocol has been briefly described in several papers, but a more recent one is,
Lin, S., Zhang, H., Zhuang, Y., Bao, T. and Gill, J. 2010. Spliced leader-based metatranscriptomic analyses lead to recognition of hidden genomic features in dinoflagellates. Proc. Natl. Acad. Sci. USA 107: 20033–20038.
Basically, we preserve the samples in Trizol (volume >10 x volume of sample), freeze at liquid nitrogen or -80C immediately. Then when you are ready to do the work, if cells/tissues do not already lyse in Trizol, remove the cells/tissue to a new container and keep the Trizol solution on ice for later use. Homogenize the cells/tissue using bead-beating or motar/pestle. After that, add the Trizol solution back. Then follow Trizol protocol to get the crude extract. Next, use Qiagen RNeasy kit to purify RNA. In doing this, do not forget to mix crude extract with ethanol following instruction of Qiagen. The volume of water to elute RNA at the end depends on the amount of material you use, but in my lab we typically begin with about 5 million cells (microalgae) or a small piece (thump tip size) of rhodophyte and elute RNA in 50 microliters (elute twice, 30 uL first, and another 20 uL second time).
We typically use 50-100 ng of RNA for cDNA synthesis in a 20-uL reaction volume, then after synthesis is done, dilute it to 200uL, and use 2 uL for regular PCR and use 4 uL for real-time PCR.
Hope this helps.
I am attaching the protocol and the paper.
Article Spliced leader–based metatranscriptomic analyses lead to rec...
Since this is my first time when I am working on RNA and expression studies, I would like to know one question?
How many uL of RNA you use in a 20-uL cDNA reaction volume.
As you wrote generally 50-100 ng of RNA you use so is it final concentration in reaction volume or it is per microliter and you use 1 microgram of RNA.
Because I am suggested by my seniors in the lab to use at least 1 micro gram of RNA for cDNA synthesis.
You do not need 1 ug of RNA for cDNA synthesis. In fact trying to use too much is not good for two reasons: 1) for algae it is not easy to get high amount of RNA and if you use 1 ug for one reaction you are going to run out of RNA quickly. This situation is different from people working on animal cells, which have high levels of RNA. 2) from algal samples, there are usually high amount of inhibitory compounds that are hard to remove, and you may not detect it by measuring A260/A280 or A260/A230. In this case, if you use too much volume of the RNA solution, you can get your subsequent PCR reaction to fail. So using more diluted RNA and smaller amount often is better. This is my experience anyway when dealing with algae.
one more thing, usually the final RNA concentration after eluting from Qiagen column is between ten to several hundreds of nanograms per microliter. I usually use no more than 5 uL for cDNA synthesis.
Thanks Lin, Your suggestion is really helpful to me as I am trying to increase the concentration of RNA from Sargassum for past 3 month but its not increasing much and now I am a bit satisfied that its not only in my case when Yield is less.. its general about algae
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