Hieff NGS™ MaxUp Human rRNA Depletion Kit (rRNA & ITS/ETS) is designed to remove rRNA and 45S ITS/ETS from human, mouse and rat total RNA based on RNase H-based workflow and to retain mRNA and other non-coding RNA. This kit is suitable for both intact and partially degraded RNA samples (e.g., FFPE RNA). Since degraded FFPE samples usually contain a higher proportion of ITS/ETS than fresh tissue samples, this kit adds probes in the Human 45S ITS/ETS region, and ITS removal can significantly increase the proportion of valid data in raw data.
|Depletion Technology||RNase H|
|Sample Type||Total RNA of human, mouse and rat|
|Final Product Type||mRNA and other non coding RNAs|
|No. of Reactions||24/96 Preps|
|Starting Material Amount||100 ng~1 μg total RNA|
|Target||Remove rRNA and 45S ITS/ETS form human total RNA|
|Components No.||Name||12257ES24 (24T)||12257ES96 (96T)|
|12257-A||Hybridization Buffer||72 μL||288 μL|
|12257-B||Probe Mix (rRNA & ITS/ETS)||48 μL||192 μL|
|12257-C||RNase H Buffer||72 μL||288 μL|
|12257-D||RNase H||48 μL||192 μL|
|12257-E||DNase I Buffer||660 μL||2×1320 μL|
|12257-F||DNase I||60 μL||240 μL|
All the components are shipped with dry ice and can be stored at -15℃ ~ -25℃ for one year.
For conventional samples, rRNA and ITS/EST can be removed by more than 98%, while for FFPE samples with integrity of about 50%, rRNA and ITS/EST can be removed by more than 95%. At the same time, FFPE with poor integrity can also be removed effectively.
Table 1. rRNA Removal specificity effect
|RNA quality||Removal scheme||Input||rRNA (%)||ITS/ETS (%)||Mapping (%)|
|293T RNA; RIN=10||remove rRNA||1μg||0.19||4.6||98.28|
|remove rRNA and ITS/ETS||1μg||0.15||0.04||98.79|
|FFPE RNA; DV200 =90%||remove rRNA||500ng||0.23||4.75||95.35|
|remove rRNA and ITS/ETS||500ng||0.21||0.02||95.42|
|FFPE RNA; DV200 =50%||remove rRNA||500ng||1.4||16.28||95.57|
|remove rRNA and ITS/ETS||500ng||0.56||0.11||95.51|
|FFPE RNA; DV200 =20%||remove rRNA||1μg||0.35||67.61||58.4|
|remove rRNA and ITS/ETS||1μg||0.79||0.84||60.35|
Note: Different human RNA samples were used to remove rRNA or rRNA and ITS/ETS, and to construct libraries with RNA library Prep kit after ITS/ETS. The proportions of rRNA and ITS/ETS in offline data were analyzed by sequencing.
Table 2. Sequencing data performance
|Sample||DV200||Kit||Clean Q20 (%)||Clean Q30 (%)||Clean GC (%)||rRNA (%)||ITS/ETS (%)||unique(%)|
 Tian S, Zhang B, He Y, et al. CRISPR-iPAS: a novel dCAS13-based method for alternative polyadenylation interference. Nucleic Acids Res. 2022;50(5):e26. doi:10.1093/nar/gkac108(IF:16.971)