Hi all,
I am pretty new in the world of RNAseq, so I apologize in advance about some silly questions I need to answer.
I am planning an experiment using LCM (Laser Capture microdissection) to dissect out neurons from Human Brain and after RNA extraction, amplification and RNAseq try to understand if there are genes (overexpressed or underexpressed) that contribute to the pathological status of a neurological disease.
To do that is very important to be fully quantitative in the RNA seq analyses.
Those are my questions :
1) May please someone confirm that is possible to run RNAseq from picograms of RNA (of course using amplification KITs to get mg of cDNA)
2) Which is the best amplification kit to use according to you ? ( I was planning to use the Ovation RNAseq V2 which looks like to be very good although very expensive...there are other valid options ?)
3) How deep the RNAseq must be to be fully quantitative ? ( I was recently discussing the possibility to run RNAseq by ILLUMINA miSeq with 12-15 millions reads per run. On the other hand, I just saw a paper (which a similar strategy I would like to use) were the authors to be "quantitative" used the ABI SOLID obtaining a total of 375 millions reads 50 bp from 4 samples.
I suspect 12 millions reads are not going to be enough to undestand if genes are over or under expressed ? There are any suggestions or thoughs about that ?
4) How much is going to cost RNAseq (45-50 millions reads per sample) with ILLUMINA Hiseq 2000 more or less ?
Thanks a lot in advice for your help.
Cheers
cherokee
I am pretty new in the world of RNAseq, so I apologize in advance about some silly questions I need to answer.
I am planning an experiment using LCM (Laser Capture microdissection) to dissect out neurons from Human Brain and after RNA extraction, amplification and RNAseq try to understand if there are genes (overexpressed or underexpressed) that contribute to the pathological status of a neurological disease.
To do that is very important to be fully quantitative in the RNA seq analyses.
Those are my questions :
1) May please someone confirm that is possible to run RNAseq from picograms of RNA (of course using amplification KITs to get mg of cDNA)
2) Which is the best amplification kit to use according to you ? ( I was planning to use the Ovation RNAseq V2 which looks like to be very good although very expensive...there are other valid options ?)
3) How deep the RNAseq must be to be fully quantitative ? ( I was recently discussing the possibility to run RNAseq by ILLUMINA miSeq with 12-15 millions reads per run. On the other hand, I just saw a paper (which a similar strategy I would like to use) were the authors to be "quantitative" used the ABI SOLID obtaining a total of 375 millions reads 50 bp from 4 samples.
I suspect 12 millions reads are not going to be enough to undestand if genes are over or under expressed ? There are any suggestions or thoughs about that ?
4) How much is going to cost RNAseq (45-50 millions reads per sample) with ILLUMINA Hiseq 2000 more or less ?
Thanks a lot in advice for your help.
Cheers
cherokee