RNA sequencing (RNA-seq) is the process of sequencing RNA via next generation sequencing. Regardless of the goal of the experiment (transcriptome sequencing, targeted transcript, non-coding RNA), RNA-seq sample preparation plays an important role in this powerful analysis method.

Learn more about RNA and transcriptome sequencing

RNA-seq sample preparation workflow

Obtaining a pure and high-quality RNA sample is critical to successful RNA-seq sample preparation. Storage of tissues from which RNA will be extracted should be carefully considered as RNA is more unstable than DNA. For long-term storage of RNA, temperatures of -80°C are often recommended to better prevent degradation and ensure the integrity of the molecules. In addition, RNA stabilization reagents can be used to help preserve RNA during storage and thawing.

How much RNA is needed for RNA-seq?

For standard RNA-seq library preparation, you should aim to start with between 100 ng to 1 µg of purified RNA. At least 500 ng purified total RNA is recommended by some core facilities.

RNA extraction for RNA-seq can be performed using a variety of methods including organic extraction (e.g., using reagents such as TRIzol), filter-based spin column methods using lysis buffers, or magnetic particle methods.

Selection guide: RNA extraction kits for RNA-seq

Best forSimple, reliable, rapid methodMicro RNA and total RNAHigh-throughput stringent applications

mRNA sequencing

High-throughput purification from FFPE tissue

Product namePureLink RNA Mini Kit mirVana miRNA Isolation KitMagMAX for Microarrays Total RNA Isolation Kit Dynabeads mRNA DIRECT Kit MagMAX FFPE DNA/RNA Ultra Kit
Starting materialBacteria, blood, cells, liquid samples (e.g., serum)Bacteria, cells, tissue, viral samplesBlood, cells, liquid samples (e.g., serum), RNA, tissueCell lysateFFPE curls
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 method/ formatSilica spin columnOrganic extraction and silica spin columnPlate-based organic extraction and magnetic beadsMagnetic bead captureMagnetic beads
Prep time20 min30 min<1 hr15 min48 min (for 96 preps)
Automation compatible?NoNoYesNoYes
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

Once extracted, it is critical that RNA be stored in a safe, RNase-free environment. There are several reagents available for the effective storage of RNA including:

Enrichment of RNA for targets or transcripts

Artistic rendition of a pool of single-strands of RNA

To maximize the number of relevant RNA-seq reads, it is often necessary to enrich the sample for specific RNA regions or targets of interest. This enrichment helps reduce the prevalence of non-transcriptome related RNA (such as ribosomal RNA) and less abundant sequences.

Read more about targeted RNA sequencing

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 ribosomal RNA. The RiboMinus method up to 99.9% of rRNA to allow greater interrogation of the less abundant transcripts.

Learn about ribosomal RNA depletion

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.

Explore kits for cDNA library construction

RNA library controls

Utilization of sequencing controls and methods for data analysis help validate the RNA-seq data from the run a well as compare results run-to-run. These mixes, designed by the External RNA Controls Consortium (ERCC) , are the accepted library controls for RNA-seq.

Best forQuantifying fold-change in expression levelsMeasuring dynamic range and LLOD of RNA-seq
 ERCC ExFold RNA Spike-In Mixes (2 x 10 µL) ERCC RNA Spike-In Mix (1 x 10 µL)
Description2 tubes containing preformulated mixes of the same 92 RNA transcripts but in different ratiosSingle tube containing a preformulated mix of 92 RNA transcripts
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

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RNA barcoding

With the evolution of sequencing platforms and reduction in cost to generate this invaluable data, the volume of RNA-seq experiments has grown rapidly. RNA barcoding allows you to keep pace by assigning unique identifiers to multiple molecules in a single library. Once barcoded, libraries can then be pooled prior to sequencing.

Get details about the Ion Xpress RNA-Seq Barcode 1-16 Kit

RNA sequencing

Once library prep is done, get ready to sequence! Ion Torrent NGS systems support a variety of RNA-seq applications with streamlined template preparation and intuitive data analysis.

Learn more about RNA-seq with Ion AmpliSeq technology

Stylesheet for Classic Wide Template adjustments

For Research Use Only. Not for use in diagnostic procedures.