Hi everyone,
please bear with me, because I am a complete beginner with regard to any form of bioinformatics and I am trying to understand the best approach to my experiment.
I am currently trying to isolate cells and sequence them for further bioinformatic analysis, more precisely RNA-Sequencing.
We have, however, had issues with purity and while some samples we looked at reached a purity of >90% after isolation (we usually validate it by use of flow cytometry), some samples of different animal genotypes did not.
This leads me to my first question:
How important is cell purity for Bulk RNA-Seq?
Which purity should be reached for and adequate, realiable analysis?
If anyone has any recommendations for papers to look into regarding that subject, I would be most grateful, because I have no idea where to start and what to consider.
Further along in the story we surmised that maybe Single Cell RNA Sequencing might be the better option in cases of lower purity.
But again, the same question arose: how relevant is cell purity for the following analysis and is there a cut-off value not to be crossed?
Finally:
How advantegeous would using both methods be?
Sure, Bulk gives a better general overview and Single Cell is more precise, but do they complement each other or is it essentially redundant information gained by doing both experiments?
And are there any disadvantages to using only SC or do both methods completement each other when low purity levels are in the question?
Thank you a lot in advance!!
Welcome to RNA-seq! It's a crazy and wild world. You will find that responses will depend a great deal on what you're aiming to achieve. So take my responses with this in mind...it depends!
Get as close to 100% as possible otherwise you'll be having to perform a set of validation experiments to ensure that any interesting findings are due to changes in your cell type of interest and not in a "contaminating" cell type. Single cell RNA-seq may be suitable here since you'll be able to get some cell type resolution and identify the populations in which the change is occurring, of course you'll still need to validate. The strenght of scRNA-seq is that you don't need to purify/enrich your population since these get resolved as part of the procedure/analysis. However, the a drawback with scRNA-seq is that you will loose a lot of low abundant transcripts, "dropout" is also a major issue. So, if you're comfortable loosing some info on potentially valuable transcripts then scRNA-seq may be the way to go. They do potentially complement each other especially because with bulk, you may get data about low expressed transcripts. But a big caveat, it all depends! You may consider identifying and collaborating with someone with expertise in RNA-seq (sample prep and data analysis) at your local institution.
Which papers? It depends. Start with papers that are answering a similar question to yours, then dig into what would be best for you study. You can consider reaching out to representative of companies like 10X Genomics and Miltenyi Biotec...that's also a good starting point. Good luck!
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