RNA Sequencing Sample Preparation | Thermo Fisher Scientific

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

Steps for RNA extraction for next generation sequencing

RNA-seq sample preparation workflow

1. RNA isolation and stabilization

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 for	Simple, reliable, rapid method	Micro RNA and total RNA	High-throughput stringent applications	mRNA sequencing	High-throughput purification from FFPE tissue
Product name	PureLink RNA Mini Kit	mirVana miRNA Isolation Kit	MagMAX for Microarrays Total RNA Isolation Kit	Dynabeads mRNA DIRECT Kit	MagMAX FFPE DNA/RNA Ultra Kit
Starting material	Bacteria, blood, cells, liquid samples (e.g., serum)	Bacteria, cells, tissue, viral samples	Blood, cells, liquid samples (e.g., serum), RNA, tissue	Cell lysate	FFPE curls
RNA types isolated for sequencing	Large RNA molecules only (mRNA and rRNA)	Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA)	Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA)	mRNA only	total RNA, microRNA, gDNA
Isolation method/ format	Silica spin column	Organic extraction and silica spin column	Plate-based organic extraction and magnetic beads	Magnetic bead capture	Magnetic beads
Prep time	20 min	30 min	<1 hr	15 min	48 min (for 96 preps)
Automation compatible?	No	No	Yes	No	Yes
Amount of starting material	10 mg to 100 mg of tissue Up to 5 x 10⁷ cells	Up to 100 mg of tissue Up to 1 x 10⁷ cells	Up to 100 mg of tissue Up to 1 x 10⁷ cells	Up to 500 ng purified Total RNA	2 x 10 micron FFPE sections
Prep size	50 preps	40 preps	96 preps	2 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:

Learn the basics of RNA extraction
Compare methods for extraction of nucleic acids from FFPE samples

2. Target RNA enrichment

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.

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 for	Quantifying fold-change in expression levels	Measuring 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)
Description	2 tubes containing preformulated mixes of the same 92 RNA transcripts but in different ratios	Single tube containing a preformulated mix of 92 RNA transcripts
Compatible sample types	Eukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNA	Eukaryotic total RNA, rRNA-depleted total RNA, or poly(A) RNA
Dynamic range	6 logs	6 logs
Volume	10 µL (2 tubes)	10 µL

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4. RNA barcoding resources

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

Resources

Stylesheet for Classic Wide Template adjustments

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