I’m a Clinical Fellow in head and neck just starting down this road. Trying to price up how much I need to compare the NEB and Illumina Small RNA kits. The rico-zero is surprisingly expensive. I see from your protocol you have halved the volumes which would help in getting the kit to go a bit further. But can I ask what may be a stupid question? – In the epicentre protocol it says the ribozero process they describe treats 1-5ug of RNA. How much RNA is your protocol optimised for? Another stupid question is regarding RNA extraction. I was going to compare the Roche miRNA FFPE kit against the Qiagen miRNA kit. Though they include a DNase step others have found a lot of contaminating DNA in their sequencing. Should I adda TURBO DNase step? Do I perform this prior to rico-zero?
Hi Neeraj,
Sorry for the delay if you are still there. I’ve been pretty bad at answering e-mails lately. Yes, Ribo-zero is very expensive, which is why I reduced the amount of input RNA. What I did worked well starting with 1 ug of RNA but I definitely could have used 0.5 ug and reduced all the Ribo-zero volumes by half and still gotten good RNA-seq libraries.
As far as DNA contamination, we just used the Qiagen RNeasy FFPE kit with the on column DNase treatment. I’m not a big Qiagen fan. I think they rely more on legacy protocols then innovation but the experiment worked. I’m not sure of the amount of DNA contamination. I guess we could have done Qubit RNA and then DNA readings on it, but whatever it was it didn’t adversely effect the RNA-seq data. With RiboZero you get a lot of non-coding RNAs which I guess someone could confuse with DNA contamination at the data analysis step. If you’re worried about DNA contamination you could purify the RNA then do a off column DNase treatement and then re-purifiy it again over another spin column, but I don’t think it’s a issue.
I’m still surprised how well the RNA-seq went from such highly degraded RNA.
Thanks so much for your reply. his is a fantastic blog! I just wanted to ask another question if that’s ok? Is it worth stepping up to Ribo-zero Gold (and I see they even have a Globin-zero kit)? Is losing mitochonrial RNA going to be worth the extra cost?I suppose it boils down to not wasting reads. We found in our first trial microRNA sequencing run that only 1-3% of reads aligned to miRbase. Though that gave us ~500,000 reads it just left us too much doubt ver the validity of the result to want to spend money doing qPCR or write it up. Have you any experience of miRNA seq? what kind of alignments did you get?
No but I imagine the protocol I used would work with a lot less RNA because I think I only ended up doing 8 cycles of PCR to amplify the library. I would check out the new Illuimina TruSeq RNA kit, which has the RiboZero included. A nice outcome of them buying Epicentre.
Encourage you to check out the new RiboGone technology. Based on hybridization too and allows a low input of 10 ng- (up to 100 ng). This would be a blessing for FFPE users!
Ethan-omics 
Hi there,
I’m a Clinical Fellow in head and neck just starting down this road. Trying to price up how much I need to compare the NEB and Illumina Small RNA kits. The rico-zero is surprisingly expensive. I see from your protocol you have halved the volumes which would help in getting the kit to go a bit further. But can I ask what may be a stupid question? – In the epicentre protocol it says the ribozero process they describe treats 1-5ug of RNA. How much RNA is your protocol optimised for? Another stupid question is regarding RNA extraction. I was going to compare the Roche miRNA FFPE kit against the Qiagen miRNA kit. Though they include a DNase step others have found a lot of contaminating DNA in their sequencing. Should I adda TURBO DNase step? Do I perform this prior to rico-zero?
Sorry for all the questions
thanks
Neeraj
Hi Neeraj,
Sorry for the delay if you are still there. I’ve been pretty bad at answering e-mails lately. Yes, Ribo-zero is very expensive, which is why I reduced the amount of input RNA. What I did worked well starting with 1 ug of RNA but I definitely could have used 0.5 ug and reduced all the Ribo-zero volumes by half and still gotten good RNA-seq libraries.
I don’t know the cost but the new Illumina TruSeq Stranded Total RNA kit uses Ribo-Zero. I just made some libraries with the TruSeq Stranded mRNA kit and they worked well which uses Oligo dT.
http://www.illumina.com/products/truseq_stranded_total_rna_sample_prep_kit.ilmn
Uses Ribo-zero.
As far as DNA contamination, we just used the Qiagen RNeasy FFPE kit with the on column DNase treatment. I’m not a big Qiagen fan. I think they rely more on legacy protocols then innovation but the experiment worked. I’m not sure of the amount of DNA contamination. I guess we could have done Qubit RNA and then DNA readings on it, but whatever it was it didn’t adversely effect the RNA-seq data. With RiboZero you get a lot of non-coding RNAs which I guess someone could confuse with DNA contamination at the data analysis step. If you’re worried about DNA contamination you could purify the RNA then do a off column DNase treatement and then re-purifiy it again over another spin column, but I don’t think it’s a issue.
I’m still surprised how well the RNA-seq went from such highly degraded RNA.
Ethan
Thanks so much for your reply. his is a fantastic blog! I just wanted to ask another question if that’s ok? Is it worth stepping up to Ribo-zero Gold (and I see they even have a Globin-zero kit)? Is losing mitochonrial RNA going to be worth the extra cost?I suppose it boils down to not wasting reads. We found in our first trial microRNA sequencing run that only 1-3% of reads aligned to miRbase. Though that gave us ~500,000 reads it just left us too much doubt ver the validity of the result to want to spend money doing qPCR or write it up. Have you any experience of miRNA seq? what kind of alignments did you get?
Hi there,
When working with FFPE have you come across a better solution under 500 ng to remove rRNA? say 100 or a 10 ng?
SG
No but I imagine the protocol I used would work with a lot less RNA because I think I only ended up doing 8 cycles of PCR to amplify the library. I would check out the new Illuimina TruSeq RNA kit, which has the RiboZero included. A nice outcome of them buying Epicentre.
Ethan
Encourage you to check out the new RiboGone technology. Based on hybridization too and allows a low input of 10 ng- (up to 100 ng). This would be a blessing for FFPE users!
Great Article. post , I loved the points . Does someone know where my business could grab a sample Ribo-Zero form to fill in ?