highlights
- Accurate miRNA profiling: A unique single adapter and circularization strategy reduces ligation bias and increases miRNA detection.
- Direct detection: Generates small RNA libraries without the need for small RNA enrichment.
- > Gel-Free: Removes adapter dimers from any input without lengthy gel excision.
The Zymo-Seq™ miRNA Library Kit is an innovative small RNA sequencing library prep kit that uses a single-adapter and circularization strategy to reduce ligation bias and provide accurate small RNA profiling from cell-free RNA (cfRNA) and total RNA. This simple, streamlined workflow minimizes hands-on time and uses gel-free removal of adapter dimers to generate stranded small RNA libraries from plasma, biofluids, cells, tissues, and more.
To prepare Zymo-Seq™ miRNA libraries, RNA is ligated to the single miRNA adapter and the ligated product is circularized. Excess adapter is blocked and removed with specialized magnetic beads that allow a completely gel-free workflow for input as low as 1 ng. The circularized miRNA-adapter product is then reverse transcribed. The resulting cDNA is amplified using Indexing PCR primers which add on Illumina-compatible adapter and unique dual index (UDI) sequences for cost-effective multiplexing. Everything needed to make sequencing-ready libraries, including indexing primers and SPRI beads, are included in the kit. It’s RNA-Seq made simple.
| Additional Components Required (Not Provided) | Molecular biology grade pure ethanol (> 95%) |
|---|---|
| Maximum Input | 200 ng of total RNA, or RNA extracted from 200 µL of plasma or serum. |
| Minimum Input | 1 ng of total RNA, or RNA extracted from 50 µL of plasma or serum. |
| Processing Time | 2 hours hands-on, 8 hours total |
| Recommended Equipment | A 96-well Aluminum Cooler Block for PCR tubes or a 96-well PCR tube rack may be placed on ice for ease of reagent assembly and maintaining samples at 4°C. To process > 12 samples at once, a multichannel pipette with low-retention tips is recommended for mixing and the timely addition of pre-aliquoted reagents. |
| Recommended Input | 100 ng of total RNA, or RNA extracted from 200 µL of plasma or serum. |
| Required Equipment | Thermal cycler, magnetic stand for 0.2 mL PCR tubes or 96-well plate, mini centrifuge, and micro centrifuge. |
| Sample Type | High integrity total RNA (RIN > 7) containing miRNA, or cell-free RNA extracted from plasma/serum samples not preserved with heparin. Small RNA enrichment is not necessary. |
| Sequencing Platform Compatibility | Libraries are compatible with all Illumina sequencing platforms. |
FAQs
The Zymo-seq miRNA Library Kit can be used with a variety of RNA samples that contain microRNA, including plasma, urine, saliva, exosomes, and total RNA extracted from various cells and tissues. Many purification methods do not adequately purify small RNA. It is critical that the RNA purification method used effectively retains the microRNA and small RNAs, such as Direct-zol Microprep, Quick-RNA Microprep, and Quick-cf-RNA Serum & Plasma Kit.
When using total RNA, we recommend using high quality RNA with a RNA Integrity Number (RIN) value of at least 7. RNA with a lower RIN value may be used but may result in over-representation of rRNA sequences due to the presence of higher levels of fragmented rRNA. This could reduce the representation of miRNAs and other small RNAs in the final library.
The total RNA may be used without size selection. RNAs larger than ~100 nt within the sample are not effectively incorporated into the final library.
Zymo-Seq miRNA libraries are compatible with any Illumina® sequencer, as well as the Element Biosciences AVITI™ system (with Element Adept Library Compatibility workflow.). This kit has not been validated on Ion Torrent®, Oxford Nanopore®, PacBio®, or other non-Illumina sequencing platforms.
Libraries may be sequenced at any length. However, it is important to note that reads longer than the typical insert size (~21nt-100nt) will require more adapter trimming through the excess sequencing cycles. For optimal results, we recommend sequencing with a minimum read length of 50 base pairs (bp) and a maximum of 75 bp and single-end sequencing
Kit Components
| Cat # | Name | Size |
|---|---|---|
| E2003 | ZymoTaq PreMix | (50 Rxns.) (2 x 625 µl) |
| E2004 | ZymoTaq PreMix | (200 Rxns.) (8 x 625 µl) |
| W1001-1 | DNase/RNase-Free Water | 1 ml |
| W1001-4 | DNase/RNase-Free Water | 4 ml |
| W1001-6 | DNase/RNase-Free Water | 6 ml |
| W1001-10 | DNase/RNase-Free Water | 10 ml |
| W1001-30 | DNase/RNase-Free Water | 30 ml |
| D4003-2-6 | DNA Wash Buffer (Concentrate) | 6 ml |
| D4003-2-48 | DNA Wash Buffer (Concentrate) | 48 ml |
| D4003-2-24 | DNA Wash Buffer (Concentrate) | 24 ml |
| D3004-4-10 | DNA Elution Buffer | 10 ml |
| D3004-4-50 | DNA Elution Buffer | 50 ml |
| 3DP-1002 | PCR Strip MagStand | 1 unit |
Zymo-Seq miRNA Library Kit
| Cat # | Name | Size |
|---|---|---|
| R3006 | Zymo-Seq miRNA Library Kit | 12 preps |
| R3007 | Zymo-Seq miRNA Library Kit | 96 preps |