RNA extraction for ciliate protozoan - (Jan/29/2008 )
Hi everyone~
currently I'm working on RNA extraction for ciliate protozoan (cryptocaryon irritans) and later I'm going to construct cDNA expression library. The problem is until now I still didn't get any result for RNA extraction. The size of the parasite is 20-30 x 50-70 um. How would I optimize the total RNA? I'm using TriReagent to extract the RNA. Is there any step that i would troubleshoot? thank you[b][font="Comic Sans MS"][/font][size="4"][/size]
How many cells are you using for extraction? Perhaps the biomass is not enough?
Add one volume of AquaRNA to Tri reagent (or Trizol), vortex to mix, then follow Tri Reagent protocol, you would get twice more RNA than Tri reagent alone, because AquaRNA irreversibly inactivates RNases.
hmm..i'm using theronts (the parasite) from 1000 cysts (1 cyst will produce approximately 200 theronts) for RNA extraction.
by the way, how could i find the AquaRNA. Does it work ok to parasite sample?
hmm..i'm using theronts (the parasite) from 1000 cysts (1 cyst will produce approximately 200 theronts) for RNA extraction.
by the way, how could i find the AquaRNA. Does it work ok to parasite sample?
AquaRNA at http://www.aquaplasmid.com/AquaRNA.html AquaRNA works for all living things tested. Here is RNA from a single tiny spider.
Actually I still think the cell number is kinda low.. Other than that you may use a more sensitive method to detect your RNA. I think I'd need more details to help further.
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)well i use trireagent and all chemicals n equipments treated with DEPC.
here i attached dat parasite's picture