Hi, I am planning to check the difference of gene expression (including coding and non-coding genes) for RNA of a time course during induced differentiation of MSC (the mesenchymal stem cells). I have performed induction of two patient's clones and two clones of normal individuals. For each of the four clones, I reserved RNA at 10 time spots (Day0, Day4, Day7,......) during the differentiation course with triplication (3 wells for each time spot of each clone). I also did a non-induced control experiment of the four clones. So I have reserved quite a lot of RNA on my hand
4 (two patient's clones and two normal clones) * 10 (time spots) * 3 (triplicates) * 2 (induced and non-induced) = 240
and sequencing all of them would be too expensive.
May I have your suggestions on how to design a RNA-seq experiment for the materials I had? Thank you very much in advance!
-
Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
-
I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...