Basic help with RNA seq

Question

[fionajcunningham]

Dear all,

I am trying to decide if I should use RNA seq to answer my research question or not and am struggling to find a basic text which helps me to understand the function of it and its applicability. I have already searched for such a text but I just want to know if there is any point in me using rna seq (single cell) to determine the amount of a particular gene in my sample - we already know there is only a small population of these cells ie Th22 cells are a small subset of Th cells.

I am trying to determine if the amount of these cells is greater in one type of patient with skin disease compared with another patient with a slightly different subtype of the disease.

Please could someone kindly recommend a text to help me to understand the basics of rna seq NOT the technical side of things but the basics of how it can be used in different scenarios.

Thanks very much for any help

Fiona

[GenoMax]

My feeling is that this would be very much dependent on the expression levels of the gene. If gene you are looking at is lowly expressed then chances of being able to see counts for it are close to nil. In best of circumstances you will likely detect 20-30% of expressed genes in a single cell experiment. At least that is my peripheral understanding.

I am trying to determine if the amount of these cells is greater in one type of patient with skin disease compared with another patient with a slightly different subtype of the disease.

That is a different question that can't be easily answered by RNAseq of any type.

[fionajcunningham]

Hi GenoMax, thank you for taking the time to reply. I understand that using bulk RNA seq the signal from cells which are a small population might be difficult to detect compared with ssRNA seq is this correct? thanks alot!Fiona

Answer 1

[jared.andrews07]

If you're interested in cell type proportions, you're generally much better off with a flow-based approach. Or even IHC, depending on cell of interest sparsity. This is often challenging to address in single cell unless you're doing whole tissue, which has its own set of issues since cell isolation/dissociation methods can bias for certain populations over others. Skin in particular is challenging to get T cells out of (from personal experience) for single cell.

[ATpoint]

In addition to that, you will need good experimental replication to show differences of cellular proportions, especially in humans that can be confounded by a lot of factors, and with single-cell technology this is going to be very expensive. Frankly, as a reviewer I would ask for FACS-based validation of this if you do it with NGS technology because all the enrichment and preparation steps in scRNA-seq can lead to all kinds of biases. I think a simple FACS-based assessment is much cheaper, more straight-forward and in this context more reliable. @OP, since you ask about NGS, is there any reason the more traditional techniques like FACS or IHC do not work here? Are there no antibodies for the particular markers you identified or are there no markers that are targetable in general?

[fionajcunningham]

hello ATpoint, this is very helpful information and I really appreciate the time you took to answer this . I am repeatedly having difficulty with staining IL22 intracellularly for FACS the fluorochromes I have tried (Multiple) don't seem to work well in the panel I am working with. I am finding it difficult to make any progress at all! My supervisor and I had good results initially but another year of experiments and the fluorochrome was poor. I will have to have another think about it. thanks Fiona.

[fionajcunningham]

thank you jared.andrews07 for taking the time to reply and provide your insights which are most helpful. I am also finding staining for intracellular IL22 extremely difficult with flow ! I hopefully will find a way forward!