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Bioanalyzer results for fragmented total RNA are continuously coming up with mean peak around 105-110nt. As a result, I suspect, I'm loosing the vast majority of the material during size selection with AMpure beads, so when reaching the library analysis I'm ending up with zero results! I'm following SOLID 4 total RNA protocol. For fragmentation I'm starting with minimal amount of rRNA depleted total RNA of at list 200ng and using RNAse III 10min for the fragmentation. Here you can see my results before and after fragmentation procedure. What I'm doing wrong? Is it possible that the initial amount of RNA is too low?
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Any ideas, guys? I'm quite desperate!
I'll be happy to hear the opinions of the experienced users
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I've never run the SOLID protocol, but for our Illumina protocols we use the NEB Next RNA fragmentation kit. It's a chemical rather than an enzymatic fragmentation. We end up with average sizes around 250 bases (customizable based on fragmentation time). We also input 200ng of rRNA depleted RNA as our starting material. Good luckComment
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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...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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