I would like to extract RNA from crustacean tissue and externally attached bacterial symbionts. I plan to use a Qiagen tissue lyser and a Qiagen RNA extraction mini kit. I would like some advice on the number and type of beads, volume of lysis buffer and beating cycles others may have used to extract animal tissue and bacterial RNA with good results. I have only a few replicates of my samples and do not want to risk doing a poor extraction. Thanks for any advice!
Essentially you are limited by the binding capacity of your column. According to Qiagen that's 100ug of RNA. They recommend no more than 30mg for tissue and 1X10^9 cells for bacteria. This should be done in 700uL of GITC-containing lysis buffer (provided in the kit). If your going to use beads I would recommend 2.5 mm ceramic beads for your tissue samples. Typically the ratio is 1:1 for beads vs sample, with 4000rpm at 2-3 cycles and short resting intervals (30sec-1min). For your bacterial cells you may want to use a smaller bead .5mm. To get the weight (approximate) you could record the weight of a micro centrifuge then pellet your bacteria in that same tube then reweigh and subtract original. That being said the lysis buffer itself should take care of the bacteria without the need for using beads. Alternatively you could use a homogenizer for your tissue samples as well. Hope this helps.
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