Hi, I am dealing with plant parasitic nematodes. I tried extracting RNA from almost 1000J2 worms with two different kits (Qiagen RNeasy Kit, Omega RNA extraction Kit) and by two different ways of cells homogenization (bead beating for 30 sec and grinding through plastic pestles). However, I am always getting very low yield of RNA (max. 10 ng/ul). My elution volume is 30ul. I need at least 500-1000ng for cDNA sysnthesis and for down stream RT-qPCR.
I will be thankful if someone could suggest me a better way to get RNA from these worms. They have a cuticle outside their body which might hinder the lysis.
Add in a proteinase K digestion step to help disrupt the cuticle. Maybe even add in using a "shredder" column like folks use for insects, plants, & other tough to disrupt tissues. I know the folks at Qiagen would be happy to help you out!
Good luck!
Trizol was working ok for me and was getting good concentrations. If the quality is still not good, try an additional ethanol precipitation step. Keep the J2s in a 1.5ml tube and with a micropestle you should be able to homogenise them thoroughly. With column-based kits you will loose lots of RNA. Hope that helps!
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