mRNA Library Prep

Lower sequencing costs for gene expression studies

The Invitrogen Collibri 3' mRNA Library Prep kits for Illumina Systems capture the 3' end of the transcript, enabling gene expression studies from as little as 2 million reads per sample, compared to 60 million reads for whole-transcriptome sequencing or 30 million reads for mRNA sequencing.

Simplified informatics, powerful insights

  • Suitable for 100 pg – 500 ng of total RNA
  • Ribosomal depletion not required
  • Multiplex up to 96 single-indexed libraries per run
  • Includes globin-depletion reagents
  • For highest success rates, tracking dyes in each reagent change color when properly mixed
  • Biologist-friendly data analysis software from Genialis allows you to explore differential gene expression

Study viral infections

 Collibri 3’ mRNA NGS Library Prep Kit contains adapters

Collibri 3’ mRNA Library Prep kits

Stranded?Yes
Reads per sample (m)2–5
Total RNA input (ng)0.1–500
Library preparation total time (hr)4.5
Sample typeTotal RNA
rRNA depletion required?No
Instrument compatibilityAll Illumina NGS systems
Recommended run length1 x 75 bp or 1 x 50 bp
Sample compatabilityTissue, FFPE, blood
Species compatibility (tissue, FFPE)
All
Species compatibility (blood) Human
Recommended applicationGene expression
  

Eliminate the need for length normalization

The protocol generates one fragment per transcript, resulting in accurate gene expression values and eliminating the need for length normalization. Sequences obtained are close to the 3’ ends of the transcripts with the majority of inserts greater than 75 bp in size. The final library size averages ≥200 bp for a recommended run length of 1 x 75 bp or 1 x 50 bp.

Cost-saving tip

Paired-end sequencing is not recommended for Collibri 3’ mRNA libraries as the quality of Read 2 would be very low due to the poly(T) stretch at the beginning of Read 2. It is recommended to reduce sequencing costs by choosing single-end sequencing when performing 3’ mRNA sequencing for gene expression studies.


Suitable for library preparation using 100 pg–500 ng of total RNA

 Ultra-low input
(100 pg–1 ng)
Low input
(1 ng–200 ng)
Standard input
(200 ng–500 ng)
Highest sensitivity of low-abundance transcripts  Yes
Suitable for high-quality total RNA?YesYesYes
Suitable for degraded RNA?May experience some loss of detectionYesYes

For accurate quantitation of input RNA, we recommend using the Qubit RNA BR Assay Kit.


Ribosomal depletion not required

The Collibri 3’ mRNA Library Prep Kit uses total RNA as an input, therefore there is no need for prior poly(A) enrichment or rRNA depletion. First-strand synthesis is initiated by oligo(dT) priming. An oligo(dT) primer containing an Illumina-compatible sequence at its 5’ end is hybridized to the RNA and reverse transcription is performed. The Collibri 3' Blood mRNA Library Prep Kit includes reagents to deplete globin to maximize the efficiency of sequencing reads. Reads mapping to globin mRNA will be reduced up to 80%.


Visual feedback at each step in the workflow

For highest success rate, tracking dyes change color at each step only when proper mixing is achieved. Lack of a color change indicates the need to pause and complete mixing prior to continuing. Visual feedback is useful to monitor the performance of robotic systems.

Video: Collibri NGS library prep kits for Illumina systems

Collibri NGS library prep kits contain unique tracking dyes in each reagent to show in real time if samples are properly mixed.

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Identify true gene expression from user effects

Variation in RNA seq expression data can be attributed to a variety of factors ranging from the quality of the starting material to the person performing the experiment. The External RNA Controls Consortium (ERCC), an ad-hoc group of academic, private, and public organizations hosted by the National Institute of Standards and Technology (NIST), developed a common set of external RNA controls to distinguish between these sources of variability and true gene expression. The controls consist of a set of unlabeled, polyadenylated transcripts designed to be added to an RNA sequencing experiment after sample isolation in order to measure against defined performance criteria.

The Invitrogen ERCC Spike-In Mixes are pre-formulated blends of 92 transcripts, derived and traceable from NIST-certified DNA plasmids. The transcripts mimic natural eukaryotic mRNAs of 250 to 2,000 nt in length. Inclusion of ERCC controls in RNA sequencing experiments establishes a standard measure for data comparison across gene expression experiments and makes it possible to measure the sensitivity (lower limit of detection) and dynamic range of an experiment.

Transcript molar ratios in ERCC Spike-In Mixes

Figure 2. Transcript molar ratios in ERCC Spike-In Mixes. The transcripts in Spike-In Mix 1 and Spike-In Mix 2 are present at defined Mix 1:Mix 2 molar concentration ratios, described by four subgroups. Each subgroup contains 23 transcripts spanning a 106-fold concentration range, with approximately the same transcript-size distribution and GC content.

Usage tip

Do not use the ERCC RNA Spike-In Mixes with highly degraded samples such as FFPE RNA. High quality ERCC RNA can outcompete lower quality RNA populations, causing ERCC RNA to be over-represented in the final library.


Biologist-friendly data analysis software

Genialis Expressions software consistently annotates and processes sequencing data at scale using preconfigured and validated pipelines. A rich suite of visualization modules allows you to follow your curiosity and, in real time, explore the data using a variety of interactive tools for differential expression analysis.

Figure 3. Analysis software designed for biologists. FASTQ files resulting from libraries generated by the Collibri 3' mRNA Library Prep kits and the Collibri 3' Blood mRNA Library Prep kits can be analyzed using Genialis Expressions software.


3' mRNA library preparation workflow

Retain coding and noncoding RNA diversity

We offer a range of Invitrogen genomic RNA extraction kits for sensitive, scalable purification from an expansive set of starting materials to help maximize process efficiency and downstream performance. This includes a broad range of kits for purifying RNA from a variety of samples including tissue, cells, blood, serum, plants, forensic samples, and more.
Automate RNA extraction using magnetic bead-based technology and KingFisher instruments to bind, wash, and elute RNA to reduce your hands-on time while maintaining high yields and excellent reproducibility.

Quantification options to scale throughput

The qPCR-based Collibri Library Quantification kit scales well for larger sample batches and is the ideal method for precious samples or clinical samples. Quantification accuracy is equivalent to the KAPA Library Quantification kit with the additional benefit of visual feedback during the quantification process.

The Qubit dsDNA HS Assay is a fluorometric assay that uses dsDNA-binding dyes in order to accurately determine NGS library concentration, and benefits from a simple workflow of just a few minutes per sample. 

Regardless of the assay that is chosen, good laboratory technique should be used in order to ensure accurate measurement of library concentrations and high-quality Illumina sequencing data.

Recommended accessories

With Applied Biosystems QuantStudio real-time PCR systems, you get true value with excellent performance, reliability, and world-class support. Our family of instruments enables you to obtain the results you need, connect and collaborate with colleagues, and achieve your research goals.

Retain coding and noncoding RNA diversity

We offer a range of Invitrogen genomic RNA extraction kits for sensitive, scalable purification from an expansive set of starting materials to help maximize process efficiency and downstream performance. This includes a broad range of kits for purifying RNA from a variety of samples including tissue, cells, blood, serum, plants, forensic samples, and more.
Automate RNA extraction using magnetic bead-based technology and KingFisher instruments to bind, wash, and elute RNA to reduce your hands-on time while maintaining high yields and excellent reproducibility.

Quantification options to scale throughput

The qPCR-based Collibri Library Quantification kit scales well for larger sample batches and is the ideal method for precious samples or clinical samples. Quantification accuracy is equivalent to the KAPA Library Quantification kit with the additional benefit of visual feedback during the quantification process.

The Qubit dsDNA HS Assay is a fluorometric assay that uses dsDNA-binding dyes in order to accurately determine NGS library concentration, and benefits from a simple workflow of just a few minutes per sample. 

Regardless of the assay that is chosen, good laboratory technique should be used in order to ensure accurate measurement of library concentrations and high-quality Illumina sequencing data.

Recommended accessories

With Applied Biosystems QuantStudio real-time PCR systems, you get true value with excellent performance, reliability, and world-class support. Our family of instruments enables you to obtain the results you need, connect and collaborate with colleagues, and achieve your research goals.


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