Small RNA Library Prep Kit for Illumina

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. 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)

Kit Specifications
Number of preps	24
Number of indices provided	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

Citations

Title	Circulating miRNAs associate with historical childhood asthma hospitalization in different serum vitamin D groups
Citation	Respiratory Research 2024.
Authors	Xiaoning Hong, Mingye Jiang, Alvin T. Kho, Anshul Tiwari, Haiyan Guo, Alberta L. Wang, Michael J. McGeachie, Scott T. Weiss, Kelan G. Tantisira & Jiang Li

Title	Metabolite signatures associated with microRNA miR-143-3p serve as drivers of poor lung function trajectories in childhood asthma
Citation	eBioMedicine 2024.
Authors	Kevin M. Mendez Sofina Begum Anshul Tiwari Rinku Sharma Qingwen Chen Rachel S. Kelly Nicole Prince Mengna Huang Priyadarshini Kachroo Su H. Chu Yulu Chen Kathleen Lee-Sarwar David I. Broadhurst Stacey N. Reinke Robert Gerszten Clary Clish Lydiana Avila Juan C. Celedón Craig E. Wheelock Scott T. Weiss Michael McGeachie j Jessica A. Lasky-Su

Title	A novel piwi-interacting RNA associates with type 2-high asthma phenotypes
Citation	ASTHMA AND LOWER AIRWAY DISEASE 2023.
Authors	Jiang Li PhD , Xiaoning Hong MS , Mingye Jiang MS , Alvin T. Kho PhD , Anshul Tiwari PhD , Alberta L. Wang MD, MS , Robert P. Chase MS , Juan C. Celedón MD, DrPH , Scott T. Weiss MD, MS , Michael J. McGeachie PhD , Kelan G. Tantisira MD, MPH

Title	Benchmarking omics-based prediction of asthma development in children
Citation	Respiratory Research 2023.
Authors	Xu-Wen Wang, Tong Wang, Darius P. Schaub, Can Chen, Zheng Sun, Shanlin Ke, Julian Hecker, Anna Maaser-Hecker, Oana A. Zeleznik, Roman Zeleznik, Augusto A. Litonjua, Dawn L. DeMeo, Jessica Lasky-Su, Edwin K. Silverman, Yang-Yu Liu & Scott T. Weiss

Title	Characterization of Extracellular Vesicle-Coupled miRNA Profiles in Seminal Plasma of Boars with Divergent Semen Quality Status
Citation	International Journal of Molecular Sciences 2023.
Authors	Notsile H. Dlamini, Tina Nyugen, Ahmed Gad, Dawit Resfaye, Shengfa F. Liao, Scott T. Willard, Peter L. Ryan and Jean M. Feugang

Title	Circulating MicroRNAs associated with Bronchodilator Response in Childhood Asthma
Citation	researchsquare 2023.
Authors	Rinku Sharma, Anshul Tiwai, Alvin T Kho, Alberta L. Wang, Upasna Srivastava, Shraddha Piparia, Brinda Desai, Richard Wong, Juan C Celedon, Stephen P Peters, Lewis J Smith, Charles G Irvin, Mario Castro, Scott T Weiss, Kelan G Tantisira, Michael J McGeachie

Title	Intrauterine Smoke Exposure, microRNA Expression during Human Lung Development, and Childhood Asthma
Citation	International journal of molecular science 2023.
Authors	Rosenberg, Lynne, Cuining Liu, Rinku Sharma, Cheyret Wood, Carrie A. Vyhlidal, Roger Gaedigk, Alvin T. Kho, John P. Ziniti, Juan C. Celedón, Kelan G. Tantisira, and et al.

Small RNA Library Prep Kit for Illumina