RNA Sequencing Sample Preparation

Next-generation RNA sequencing has transformed gene expression profiling and other RNA expression analysis studies. With an increasing number of researchers employing next-generation sequencing, and with the evolution of sequencing platforms, it is essential to use robust and streamlined methods for sample preparation. The best next-generation sequencing data begin with optimal samples.

Learn more about RNA & Transcriptome Sequencing

RNA Isolation

Although many methods are available for purifying RNA from any particular source, obtaining RNA of the highest purity is the best assurance that the downstream manipulations will be successful. Choose from various formats, including PureLink, mirVana, RecoverAll, and MagMAX kits to isolate total RNA, then enrich for the subpopulation of interest.

 Order nowOrder nowOrder nowOrder nowOrder now
 Simple, reliable, rapid method30 min isolation from most samplesHTP format, ideal for stringent applicationsBest for next-generation sequencing of mRNAHigh-throughput purification of FFPE samples
 PureLink RNA Mini Kit mirVana miRNA Isolation Kit (with or without phenol) MagMAX for Microarrays Total RNA Isolation Kit Dynabeads mRNA DIRECT Micro Kit MagMAX FFPE Total Nucleic Acid Isolation Kit
RNA types isolated for sequencingLarge RNA molecules only (mRNA and rRNA)Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA)Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA)mRNA onlytotal RNA, microRNA, gDNA
Isolation methodFast, convenient silica columnHighest purity and convenience; includes organic extraction and silica columnScalable, flexible format for highest purity; includes organic extraction and magnetic beadsFast magnetic bead capture directly from microsized samplesXylene-free, scalable, flexible format with magnetic beads
Prep time20 min30 min<1 hr15 min3 hr (for 96 preps)
Amount of starting material10 mg to 100 mg of tissue Up to 5 x 107 cellsUp to 100 mg of tissue Up to 1 x 107 cellsUp to 100 mg of tissue Up to 1 x 107 cellsUp to 500 ng purified Total RNA2 x 10 micron FFPE sections
Prep size50 preps40 preps96 preps2 mL oligo dT beads (sufficient for 100 mRNA preps)96 preps

RNA Enrichment

To maximize the effectiveness of sequencing reagents, it is often necessary to enrich the sample for specific regions or targets of interest. This enrichment helps eliminate nonspecific artifacts, making it possible to better analyze smaller changes in genomes or transcriptomes and less abundant sequences. Ambion RiboMinus technology is designed to enrich the whole spectrum of RNA transcripts, regardless of their polyadenylation status or the presence of a 5' cap, by selectively depleting rRNA. The RiboMinus method removes the vast majority of rRNA (up to 99.9%) to allow greater interrogation of the less abundant transcripts.

Learn more about RiboMinus technology

 Order nowOrder now
 A novel and efficient method utilizing Locked Nucleic Acids (LNA) to remove up to 99% rRNAA novel and efficient method utilizing Locked Nucleic Acids (LNA) to remove up to 99% rRNA
 RiboMinus Eukaryote Kit for RNA-Seq RiboMinus Plant Kit for RNA-Seq
PlatformDiagnosticDiagnostic
Starting material1–10 µg total RNA 100–500 ng total RNA (low-input protocol)1–10 µg total RNA
RiboMinus RNA yield1–2 µg from 10 µg human total RNA ~100 ng from 500 ng human total RNA (low-input protocol)1–2 µg from 10 µg human total RNA
Probe specificityEukaryoticPlant
Cat. No.A1083708 - 8 prepsA1083808 - 8 preps

RNA Library Construction

Library construction, template preparation, and sequencing are at the heart of the RNA sequencing workflow. Utilization of sequencing controls and methods for data analysis complete the workflow and provide useful annotated data. Generate cDNA by reverse transcription from adaptors ligated to the ends of RNA, then amplify the cDNA using primers complementary to the adaptors, and purify it.

 Order nowOrder nowOrder now
 Prepare representative cDNA libraries for strand-specific RNA sequencingA Great Solution for NGSThe Ultimate Solution for NGS
 Ion Total RNA-Seq Kit v2 SOLiD Total RNA-Seq KitAmbion RNA-Seq Library Construction Kit
PlatformIon Torrent PGMSOLiD 5500 AnalyzerIllumina
RNA input typerRNA-depleted total RNA, poly(A) RNA, or miRNATotal RNA, rRNA-depleted total RNA, poly(A) RNA, or miRNATotal RNA, rRNA-depleted total RNA, poly(A) RNA, or miRNA
RNA input range Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome: 100 ng total RNA (~1 ng poly(A) or ~25 ng rRNA-RNA
Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome:
100 ng poly(A), 200 ng
Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome:
100 ng poly(A) or 200 ng rRNA-depleted total RNA, or 200 ng total RNA
Barcodes available Yes96No
Cat. No.4475936 - 12 reaction kit4445374 - 12 reaction kit4454073 - 12 reaction kit

 

RNA Library Controls


Library construction, template preparation, and sequencing are at the heart of the sequencing workflow. Utilization of sequencing controls and methods for data analysis complete the workflow and provide useful annotated data.

Learn more about ERCC Controls

 Order nowOrder now
 2 tubes containing preformulated mixes of the same 92 RNA transcripts but in different ratios, for measuring dynamic range, assessing bias, and confirming expression fold changesSingle tube containing a preformulated mix of 92 RNA transcripts for measuring dynamic range and assessing bias
 ERCC ExFold RNA Spike-In Mixes (2 x 10 µL) ERCC RNA Spike-In Mix (1 x 10 µL)
Top Seller  
Compatible sample typesEukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNAEukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNA
Dynamic range6 logs6 logs
Volume10 µL (2 tubes)10 µL
Number of reactionsSpike-in controls diluted based on sample type and concentrationSpike-in controls diluted based on sample type and concentration

RNA Barcoding

Sets of 16 barcodes are available for use with the SOLiD Total RNA-Seq Kit or the SOLiD SAGE Kit with Barcoding Adaptor Module. Barcodes cost-effectively increase the number of samples that can be analyzed in a single sequencing run. Barcodes assign a unique identifier to templated beads made from a single library. Once assigned, the RNA libraries can be pooled and sequenced.

RNA Isolation

Although many methods are available for purifying RNA from any particular source, obtaining RNA of the highest purity is the best assurance that the downstream manipulations will be successful. Choose from various formats, including PureLink, mirVana, RecoverAll, and MagMAX kits to isolate total RNA, then enrich for the subpopulation of interest.

 Order nowOrder nowOrder nowOrder nowOrder now
 Simple, reliable, rapid method30 min isolation from most samplesHTP format, ideal for stringent applicationsBest for next-generation sequencing of mRNAHigh-throughput purification of FFPE samples
 PureLink RNA Mini Kit mirVana miRNA Isolation Kit (with or without phenol) MagMAX for Microarrays Total RNA Isolation Kit Dynabeads mRNA DIRECT Micro Kit MagMAX FFPE Total Nucleic Acid Isolation Kit
RNA types isolated for sequencingLarge RNA molecules only (mRNA and rRNA)Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA)Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA)mRNA onlytotal RNA, microRNA, gDNA
Isolation methodFast, convenient silica columnHighest purity and convenience; includes organic extraction and silica columnScalable, flexible format for highest purity; includes organic extraction and magnetic beadsFast magnetic bead capture directly from microsized samplesXylene-free, scalable, flexible format with magnetic beads
Prep time20 min30 min<1 hr15 min3 hr (for 96 preps)
Amount of starting material10 mg to 100 mg of tissue Up to 5 x 107 cellsUp to 100 mg of tissue Up to 1 x 107 cellsUp to 100 mg of tissue Up to 1 x 107 cellsUp to 500 ng purified Total RNA2 x 10 micron FFPE sections
Prep size50 preps40 preps96 preps2 mL oligo dT beads (sufficient for 100 mRNA preps)96 preps

RNA Enrichment

To maximize the effectiveness of sequencing reagents, it is often necessary to enrich the sample for specific regions or targets of interest. This enrichment helps eliminate nonspecific artifacts, making it possible to better analyze smaller changes in genomes or transcriptomes and less abundant sequences. Ambion RiboMinus technology is designed to enrich the whole spectrum of RNA transcripts, regardless of their polyadenylation status or the presence of a 5' cap, by selectively depleting rRNA. The RiboMinus method removes the vast majority of rRNA (up to 99.9%) to allow greater interrogation of the less abundant transcripts.

Learn more about RiboMinus technology

 Order nowOrder now
 A novel and efficient method utilizing Locked Nucleic Acids (LNA) to remove up to 99% rRNAA novel and efficient method utilizing Locked Nucleic Acids (LNA) to remove up to 99% rRNA
 RiboMinus Eukaryote Kit for RNA-Seq RiboMinus Plant Kit for RNA-Seq
PlatformDiagnosticDiagnostic
Starting material1–10 µg total RNA 100–500 ng total RNA (low-input protocol)1–10 µg total RNA
RiboMinus RNA yield1–2 µg from 10 µg human total RNA ~100 ng from 500 ng human total RNA (low-input protocol)1–2 µg from 10 µg human total RNA
Probe specificityEukaryoticPlant
Cat. No.A1083708 - 8 prepsA1083808 - 8 preps

RNA Library Construction

Library construction, template preparation, and sequencing are at the heart of the RNA sequencing workflow. Utilization of sequencing controls and methods for data analysis complete the workflow and provide useful annotated data. Generate cDNA by reverse transcription from adaptors ligated to the ends of RNA, then amplify the cDNA using primers complementary to the adaptors, and purify it.

 Order nowOrder nowOrder now
 Prepare representative cDNA libraries for strand-specific RNA sequencingA Great Solution for NGSThe Ultimate Solution for NGS
 Ion Total RNA-Seq Kit v2 SOLiD Total RNA-Seq KitAmbion RNA-Seq Library Construction Kit
PlatformIon Torrent PGMSOLiD 5500 AnalyzerIllumina
RNA input typerRNA-depleted total RNA, poly(A) RNA, or miRNATotal RNA, rRNA-depleted total RNA, poly(A) RNA, or miRNATotal RNA, rRNA-depleted total RNA, poly(A) RNA, or miRNA
RNA input range Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome: 100 ng total RNA (~1 ng poly(A) or ~25 ng rRNA-RNA
Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome:
100 ng poly(A), 200 ng
Small RNA: total RNA containing 1–100 ng of miRNA

Whole transcriptome:
100 ng poly(A) or 200 ng rRNA-depleted total RNA, or 200 ng total RNA
Barcodes available Yes96No
Cat. No.4475936 - 12 reaction kit4445374 - 12 reaction kit4454073 - 12 reaction kit

 

RNA Library Controls


Library construction, template preparation, and sequencing are at the heart of the sequencing workflow. Utilization of sequencing controls and methods for data analysis complete the workflow and provide useful annotated data.

Learn more about ERCC Controls

 Order nowOrder now
 2 tubes containing preformulated mixes of the same 92 RNA transcripts but in different ratios, for measuring dynamic range, assessing bias, and confirming expression fold changesSingle tube containing a preformulated mix of 92 RNA transcripts for measuring dynamic range and assessing bias
 ERCC ExFold RNA Spike-In Mixes (2 x 10 µL) ERCC RNA Spike-In Mix (1 x 10 µL)
Top Seller  
Compatible sample typesEukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNAEukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNA
Dynamic range6 logs6 logs
Volume10 µL (2 tubes)10 µL
Number of reactionsSpike-in controls diluted based on sample type and concentrationSpike-in controls diluted based on sample type and concentration

RNA Barcoding

Sets of 16 barcodes are available for use with the SOLiD Total RNA-Seq Kit or the SOLiD SAGE Kit with Barcoding Adaptor Module. Barcodes cost-effectively increase the number of samples that can be analyzed in a single sequencing run. Barcodes assign a unique identifier to templated beads made from a single library. Once assigned, the RNA libraries can be pooled and sequenced.

RNA Sequencing

No matter which platform you choose to suit your needs, we continue to support the evolving needs of this field with gold-standard instrumentation, innovative next-generation technology, optimized best-in-class reagents, consumables, analysis software, applications and world-class technical support.

Next-Generation (SOLiD)

Next-Generation (SOLiD) 
Sequence right here, right now—minimizing wait time and costs for wasted reagents.

Semiconductor (Ion Torrent) Sequencing

Semiconductor (Ion Torrent) Sequencing 
Sequence from your benchtop with Ion Torrent revolutionary "on-chip" technology.

RNA & Transcriptome Sequencing

RNA & Transcriptome Sequencing 
Choose the correct technology for your specific RNA application.