RiboZero won't work?

[Neiltje]

Hi,

I'm trying to make a library of RNA following the ScriptSeq protocol. Everything works just fine except the RiboZero depletion of rRNA. I'm using young leafs of rice. An input of 2.5 µg of sample, 8 µl of removal solution.
I have to do this step twice and even then, I don't get rid of all the rRNA. I still have some nice peaks on the bioanalyzer.

[kwaraska]

I know many kits have very specific maximums and just increasing the volumes doesn't always work.

I know Epicentre has a max of 1ug and over that causes incomplete reduction.

Not familiar with Script Seq but I would suggest checking the maximum amount and doing multiple replicates if you are over the maximum.

[Neiltje]

I know many kits have very specific maximums and just increasing the volumes doesn't always work.

I know Epicentre has a max of 1ug and over that causes incomplete reduction.

Not familiar with Script Seq but I would suggest checking the maximum amount and doing multiple replicates if you are over the maximum.

According to their protocol the input should be between 1 and 5 µg. I'm using standard 2.5 µg so that cannot be an issue

[Olaf Blue]

Hi Neiltje, Which Ribo-Zero kit are you using, and what is the sample type (human/bacteria/yeast/plant)? RZ can use up to 5 µg input total RNA, and depending upon the Ribo-Zero kit used, as little as 100 ng.

[Neiltje]

Hi Neiltje, Which Ribo-Zero kit are you using, and what is the sample type (human/bacteria/yeast/plant)? RZ can use up to 5 µg input total RNA, and depending upon the Ribo-Zero kit used, as little as 100 ng.

I'm using the RiboZero plant leaf on young leafs of rice. I think it just doesn't work well on this material because the last time I tried, I also took a sample of Canola and that worked fine

[Olaf Blue]

Variations in rRNA sequences even between some species can change the outcome. Do you have the rRNA sequences for your strain of rice?