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.
Steps for RNA extraction for next generation 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 for||Simple, reliable, rapid method||Micro RNA and total RNA||High-throughput stringent applications|
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)|
|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:
Enrichment of RNA for targets or transcripts
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.
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.
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.
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|
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.
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.
For Research Use Only. Not for use in diagnostic procedures.