The key to successful single-cell RNA extraction and NGS
RNA sequencing (RNA-Seq) has facilitated transcriptomics, which is the study of the entire RNA complement in a sample. It offers a snapshot of genes expressed within a particular tissue, cell type or single cell. Comparison of cell populations under differing environmental conditions can provide information on changes in gene expression permitting a better understanding of genomics, cellular regulation and the overall function of an organism or tumour microenvironment.
RNA-Seq strategies generally use next generation sequencing technology (NGS) to analyse the transcriptome. These technologies offer an advantage over other approaches such as quantitative or real-time reverse-transcriptase PCR (RT-qPCR) or microarrays which are limited to exploring known genes for which markers can be generated. It is a flexible application, permitting analysis of the whole genome, including rare and unknown sequences, offering a glimpse into the big picture. It can however also be focused on specific subsets of genes depending on how the experiment is designed.
More recent advances have established the technique of single-cell RNA-Seq. This technique permits analysis at such a granular level, subtle but functional changes in expression within a specific cell type can be observed that would remain hidden within a larger mixed cell population. Analysis of the specific as opposed to the average expression of a gene is therefore possible. Single-cell approaches will improve our understanding of tissues and how they are formed by a complex network of distinct single cells, coordinated to achieve a specific function.
Single-cell RNA-Seq can be used to:
• Identify novel subpopulations of cells
• Compare cell types from samples across different treatments
• Map gene expression profiles for novel cell subpopulations
• Identify altered states as a result of drug or conditional challenge
Common and key to all single-cell approaches whether it is RNA-Seq or RT-qPCR however, is the requirement to first isolate high-quality RNA from single-cell samples. LubioScience, through their relationships with Norgen Biotek Corp and IDT (Integrated DNA Technologies) have the solution for both RNA isolation and purification from single cells and NGS RNA library preparation, making them your partner for single cell RNA analysis.
Single cell RNA Purification
Recent years have seen the development of highly sensitive cell separation and detection techniques such as Laser-Captured Microdissection (LCM) and fluorescence-activated cell sorting (FACS). These techniques enable isolation of small populations of tissue and single cell respectively. New single cell amplification technologies such as whole genome amplification and increases in detection limits have permitted the use of RT-qPCR, microarrays and RNA sequencing at single cell resolution. However, despite technology advancements, the number of products available for RNA purification remain limited, with most still focused on preparation from cell lysates.
The single cell RNA purification kit from Norgen Biotek Corp however, offers a rapid and sensitive method for the isolation and purification of total RNA from small input quantities of animal cells, sorted cells, and microdissected samples including LCM. With recovery of RNA from single cell to 2 x 105 quantities, the kit purifies all sizes of RNA from large mRNA to small interfering RNA (siRNA).
| Single Cell RNA Purification Kit Benefits | |
|---|---|
| Small elution volumes | The micro spin-column allows a small elution volume of as little as 8 ul. |
| Isolate a diversity of RNA species | All RNA species can be isolated from large mRNA and ribosomal RNA down to microRNA (miRNA) and small interfering RNA (siRNA) without compromising total yield. |
| Fast and easy processing | Rapid spin-column format allows the processing of 10 samples in as little as 20 minutes. |
| Ideal for small samples | Isolate total RNA from small input amounts of cultured animal cells, sorted cells, and microdissected samples, including LCM. Total RNA has been isolated and detected from single animal cells. |
| Recovered RNA is suitable for downstream applications | Purified RNA can be used in a number of downstream applications: • Real-time PCR • Reverse transcription PCR Northern blotting • RNAse protection and primer extension • Expression array analysis requiring intact RNA |
NGS RNA Library Preparation
The applications of RNA-Seq are various offering us a better understanding of genomics, diseases states, cellular regulation and overall organism function:
• Gene expression profiling
• Whole transcriptome research
• Alternative splicing isoform identification
• Rare and novel transcript discovery
• Research on gene fusions, isoforms and structural variants
• Allele-specific expression studies
• Research on diseases associated SNVs
• Total RNA + hybridization capture enrichment experiments
All RNA-Seq experiments begin with library preparation and via IDT, LubioScience is able to offer a variety of tools including:
xGen Broad-Range RNA Library Prep Kit.
For low inputs of RNA (~100 pg mRNA or ~10 ng total RNA), this kit allows you to deepen your rare and novel transcript discovery. Offering high mapping rates, greater gene and transcript detection numbers and consistent performance across a broad range of inputs.
xGen RNA Library Kit.
For higher inputs of RNA (>5 ng mRNA or >100 ng total RNA), this kit allows you to accelerate your gene expression profiling studies. Offering an expedited ~3.5 hour workflow and consistent performance across inputs.These kits off fast NGS transcriptomics research workflow with comprehensive transcript coverage and NGS data quality from a broad range of input quantities for Illumina® sequencing platforms. Leveraging proprietary AdaptaseTM technology, these RNA library prep kits enable stranded RNA library construction directly from 1st strand cDNA without the requirement for 2nd strand cDNA synthesis and degradation, or template-switching methods. The kits are compatible with manual and automated workflows, as well as upstream and downstream enrichment and depletion methods. They support a variety of indexing options in research studies.
| xGen Broad-Range RNA Library Prep Kit | xGen RNA Library Kit |
|---|---|
| Comprehensive data - for mapping rates, gene identification & transcript coverage | Reliable functionality – maintains ligation efficiency at all supported inputs with no adapter titration required |
| Consistent results - from 10 ng to 1 ug total RNA or 100 pg to 100 ng mRNA input | Indexing – combinatorial dual indexing up to 96-plex, unique dual indexing up to 1536-plex and enzymatic library normalization available. |
| Consistent libraries – minimal adapter dimers = no adapter titration for supported inputs | Automation-friendly |
| Save time and cost – From RNA to library in 4.5 hours with a wide variety of index options from 1536 UDI primer pairs to 96 CDI primer pairs | Save time and cost – results in as little as 3.5 hours. Process more samples in less time to increase your data output. |
| Workflow – designed for easy automation | Workflow – supports many workflows with indexing options, automation, and Normalase™ Technology |
Kit specifications
| Feature | Specification - xGen Broad-Range RNA Library Prep Kit | Specification - xGen RNA Library Kit | Benefit |
|---|---|---|---|
| Input quantity | 10 ng–1 µg total RNA 100 pg–100 ng mRNA | 100 ng to 1 μg total RNA 5 ng to 100 ng mRNA | Supports a wide input range Consistent library output |
| RNA types supported | Poly(A)-enriched mRNA Ribo-depleted RNA Total RNA | Supports most RNA applications | |
| Technology | Adaptase tailing and ligation of 1st strand cDNA | No 2nd strand cDNA No adapter titration Fewer dimers and duplicates detected in internal research studies Maintains strandedness (≥97%) Higher mapping, transcript detection | |
| Workflow time | 4.5 hours | 3.5 hours | Less hands-on time |
| Kit reaction sizes | 4x96, 96 and 16 | Evaluation and adoption | |
| Components provided | Fragmentation module RT module Library prep Polymerase | Complete solution for processing total, enriched, or depleted RNA from transcript to library | |
| Indexing options | Combinatorial dual Unique dual Normalase compatible | Flexible for different sequencers, workflows, and applications | |
| Multiplexing capacity | Up to 1536 libraries | Save sequencing costs | |
| Automation | Compatible with liquid handlers Custom packaging available | Supports high-throughput applications | |
Single-cell RNA seq Services:
The easiest way to get your single-cell RNA seq results is by using LubioScience’s NGS single-cell RNA seq service services.
The service includes:
- Cell preparation
- Sample processing
- Library generation
- Sequencing
- Bioinformatic analysis
Contact us for a fast and personal consultation at
SELECTED PRODUCTS AND FURTHER INFORMATION
| Cat-No. | Item | Size | Price (CHF) |
|---|---|---|---|
| 51800 | Single Cell RNA Purification Kit | 50 preparations | 545.00 |
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