Is rRNA depletion or mRNA enrichment more suitable for RNA library preparation kits?

rRNA depletion and mRNA enrichment are not absolutely a matter of which is better: if you only care about differential expression of coding genes, the polyA method is usually lower in cost and leaves extremely low rRNA contamination; but since you have already found that noncoding information is lost, switching to rRNA depletion is a reasonable choice. 200 ng of total RNA is not low, but you should note the RNA loss after rRNA depletion, so the RNA actually entering library preparation may be only a few tens of nanograms; therefore, the library preparation kit should preferably have low-input compatibility. Dimensions for supplier comparison: probe species coverage, depletion step time, whether it includes a strand-specific dUTP module, small-package sizes, and whether a trial can be requested. I suggest using samples from the same treatment group to run a small 3 vs 3 pilot test, focusing on the number of significant genes in differential expression analysis and inter-sample reproducibility; whether rRNA contamination is high is only one of the QC points.

<p>For differential expression analysis, if you want to simultaneously cover non-coding RNAs such as lncRNA and circRNA, rRNA depletion is recommended. 200 ng of total RNA is sufficient. The level of rRNA carryover mainly depends on probe species matching and RNA integrity. I typically take a sample after depletion and run it on an RNA Pico or standard RNA 6000 assay to check whether the 18S/28S peak heights are significantly reduced; after sequencing, I then examine the proportion of reads mapping to rRNA, which is usually</p>

We previously did differential expression in tumor tissues and switched from polyA to rRNA depletion, mainly because we wanted to capture lncRNAs. For library preparation with 200 ng total RNA using rRNA depletion, I have not encountered very high rRNA residual levels, provided that freshly extracted samples with RIN > 7 are used. If the samples are degraded, with RIN 4-6, the rRNA depletion efficiency depends more on the kit's depletion probes, and the polyA method may instead lose a lot of information. If you are concerned about high residual levels, I suggest that when comparing kits, ask the manufacturer to provide the residual rate range from actual samples, rather than only looking at promotional materials. Small-size trial packs can be requested, and it is advisable to compare two vendors by preparing one library each from the same batch of 200 ng RNA and submitting them for shallow sequencing to assess gene detection numbers, rRNA proportion, and strand specificity. This is more reliable than just reading the instructions.