All the reagents and components required to generate small RNA libraries to be used for next-generation sequencing on an Illumina platform
For research use only and NOT intended for in vitro diagnostics.
Features and Benefits
- Optimized for low input RNA, especially from bodily fluids such as plasma or serum at 1ng of RNA
- Simple and quick workflow: library could be prepared in less than 5 hours
- No gel purification for selected types of samples
- Protocol optimized for RNA isolated from different types of input, including liquid biopsies (blood, plasma, serum and urine)
- Complements Norgen's Best-in-Class Total RNA (including microRNA) Purification Technology
The Small RNA Library Prep Kit for Illumina consists of all the reagents and components required to generate small RNA libraries to be used for next-generation sequencing on an Illumina platform. All molecular reagents including adaptors, primers, enzyme mixes and buffers are provided. A purification module is also provided for rapid purification of nucleic acid products generated at various steps of the workflow. The purification module utilizes Norgen’s patent resin technology which enhances recovery of desired library intermediates or final products. The library prep workflow could be used for different forms of input including purified total RNA or enriched small RNA, as well as RNA from low content inputs such as plasma, serum and urine.
Workflow
Details
Supporting Data
Figure 1. Small RNA Library Prep Kit for Illumina.
An example of a purified small RNA library on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA Chip. The library was prepared using a mixture of synthetic microRNAs as an input. A single peak of ~ 141 bp was obtained and could be used directly for analysis on an Illumina next-generation sequencing platform.
Figure 2. An example of a small RNA library on resolved on a 6% Novex® TBE PAGE gel.
The library containing microRNAs resolved at around 140 bp and migrated just below the 150 bp marker of the provided NGS MW Ladder. The NGS Control Ladder contains two bands for alignment during band excision. The top band co-migrate with the expected size of library construct containing mainly microRNAs (~140 bp). The bottom band co-migrate with the expected size of an adaptor-adaptor-ligation product (~ 120 bp) that should be excluded.
Figure 3. Superb reproducibility of constructing small RNA (microRNA) sequencing libraries from liquid biopsies samples with low RNA abundance.
Total RNA was prepared from 200 µL of EDTA-plasma using Norgen's Plasma/Serum RNA Purification Mini Kit (Cat. 55000). Small RNA libraries (four replicates) were prepared from purified RNA using Norgen’s Small RNA Library Prep Kit for Illumina (Cat. 63600) without the use of gel purification (prep time of ~ 5 hours). The resulting libraries were sequenced on Illumina's MiSeq system using v3 chemistry. Reads were mapped to microRNAs and normalized to reads per million. Pairwise-comparisons of microRNA expression among all replicates were performed. High correlations (or reproducibility) were observed among all replicates with an average of over 99%.
Figure 4. Combining true total RNA purification technology with robust production of small RNA (microRNA) sequencing libraries from RNA purified from whole blood and preserved blood.
Total RNA was prepared from either whole blood or collected in Paxgene™ or Tempus™ blood tubes using Norgen’s Total RNA Purification Plus Kit (Cat. 48300), Preserved Blood RNA Purification Kit II (for use with Paxgene™ Blood RNA Tubes) (Cat. 43500) or Preserved Blood RNA Purification Kit I (for use with Tempus™ Blood RNA Tubes) (Cat. 43400), respectively, in duplicates. Small RNA libraries were prepared from purified RNA using Norgen's Small RNA Library Prep Kit for Illumina (Cat. 63600). The resulting libraries were sequenced on Illumina's MiSeq system using v3 chemistry. Reads were mapped to microRNAs and normalized to reads per million. Pairwise-comparisons of microRNA expression among all replicates were performed. High correlations (or reproducibility) were observed among all replicates of each blood tube with an average of over 99% (Panel A). Moreover, all three types of blood samples yielded similar numbers of microRNA detected (Panel B) with highly correlated expression profiles (Panel C)
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Kit Specifications
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| Number of preps |
6 or 24
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| Number of indices provided | 6 (for 6 preps) or 12 (for 24 preps, indexes 1-24 or 25-48) |
| Time required to complete 6 libraries | As little as 5 hours |
| Input RNA required | As little as 50 pg |
Storage Conditions and Product Stability
Some components require storage at -20°C, 4°C or room temperature. See individual components and box labels for storage conditions.
| Step | Component | Cat. 64600 (24 preps) |
|---|---|---|
| 3' AdaptorLigation to Template RNA | 3' Adaptor | 30 µL |
| 3' Adaptor Ligation Master Mix | 320 µL | |
| T4 RNA Ligase 2 (Truncated) | 35 µL | |
| 5' Adaptor Ligation | 5' Adaptor | 30 µL |
| 5' Adaptor Ligation Master Mix | 320 µL | |
| T4 RNA Ligase 1 | 35 µL | |
| cDNA Synthesis from Ligated RNA Product | Reverse Primer | 30 µL |
| cDNA Synthesis Master Mix | 220 µL | |
| TruScript ReverseTranscriptase | 35 µL | |
| PCR Amplification | 2x NGS PCR Master Mix | 1.32 µL |
| PCR Reverse Primer | 81 µL | |
| Forward Index Primer | Included in Small RNA Library Prep Forward Index Primers (# 64640 or # 64610) | |
| Size Selection | NGS MW Ladder | 50 µL |
| NGS Control Ladder | 50 µL | |
| Loading Dye | 300 µL | |
| Nuclease-Free Water | 1.25 µL |
| Component | Cat. 63500 (75 preps) |
|---|---|
| Buffer RL | 40 mL |
| Wash Solution A | 38 mL (Reconstituted to 128 mL) |
| Elution Solution A | 6 mL |
| Columns | 75 |
| Gel Filtration Columns | 24 |
| Collection Tubes | 75 |
| Elution Tubes | 75 |
| Product Insert | 1 |
