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I am doing RNA-Seq, of mRNA by polyA bead selection. In the protocol I am using, it says to heat treat the mRNA for fragmentation. It does heat treatments at 94C at varying time lengths for varying size fragments. It then says to transfer the heated samples to a magnet so that the hot beads bind to the magnet. I wasn't watching the temperature drop back down on my thermocycler and by the time I did, the thermocycler was at 42C. I quickly transferred my samples to the magnet. But since the temperature dropped from 94C to 42C, will I lose yield of my mRNA? Or what kinds of problems will I run into? Is anyone familiar with different mRNA fragmentation treatments? Is 94C necessary?
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The beads need to be magnetized while still hot so that the mRNA fragments containing poly(A) tails or internal poly(A) stretches will not have a chance to re-hybridize to the oligo d(T) beads. So in the situation you described, I think the biggest problem will be lower representation of these fragments in your final library. Re-heating the beads to 80 degrees and then placing them immediately on the magnet should allow recovery of these fragments without further fragmentation of the rest of the material.
I believe the temperature used for fragmentation depends on the divalent cation used in the fragmentation buffer- most library prep kits use magnesium (which is conveniently already present in RT buffer) so 94 degrees is the recommended temperature, whereas zinc-based fragmentation, such as that sold by Ambion, is performed at 70 degrees.
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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
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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...
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