Next-Generation Sequencing (NGS) has transformed the way we study gene expression, with mRNA sequencing (mRNA-Seq) becoming a cornerstone in transcriptomics research. Whether you’re new to NGS or looking to sharpen your workflow, this guide walks you through every step of mRNA library preparation—from purification to common pitfalls to avoid.

What is mRNA-Seq and Why Does It Matter?

mRNA-Seq allows researchers to capture the transcriptome, providing insights into gene expression, alternative splicing, and novel transcript discovery. It’s a powerful tool in cancer research, developmental biology, and personalized medicine.

Step 1: mRNA Purification

Why Purify mRNA?

Total RNA contains ribosomal RNA (rRNA), transfer RNA (tRNA), and other non-coding RNAs. For accurate mRNA-Seq, you need to enrich for mRNA, typically via:

• Oligo(dT) magnetic beads: Capture poly-A tails of mRNA.
• rRNA depletion kits: Useful for degraded samples or species without polyadenylated mRNA.

Figure 1. Pie chart showing the distribution of different RNA types in total RNA extracted from human tissues or cells.

Poly(A) Purification: A Key Step for Eukaryotic mRNA Enrichment

Most eukaryotic mRNAs have a poly(A) tail at their 3’ end, which makes them different from prokaryotic mRNAs. This poly(A) tail allows for easy and selective purification of mRNA.

The most common method is using Oligo(dT) magnetic beads to specifically bind and capture mRNA from total RNA (see Figure 2). This approach is simple, efficient, and widely used.

Another option is to use Oligo(dT) primers during reverse transcription to target mRNA. However, this method is less specific and not as efficient, so magnetic bead-based purification is preferred in most cases.

 Figure 2. Oligo(dT) magnetic beads specifically bind to the poly(A) tail of eukaryotic mRNA.

rRNA Removal Methods: When Poly(A) Selection Isn't an Option

For samples that lack a poly(A) tail—such as prokaryotic RNA or degraded total RNA (e.g., FFPE samples)—poly(A)-based enrichment is not possible. In these cases, rRNA removal is used to enrich for mRNA.

Category	Probe Hybridization (Ribo-Zero)	RNase H Digestion(Most commonly used)	One-Step rRNA Block (RT Blockers)	Magnetic Bead-based rRNA Removal
Principle	Species-specific probes hybridize with rRNA, removed via streptavidin beads.	DNA probes + RNase H digest rRNA in DNA-RNA hybrids.	Blocker probes prevent reverse transcription of rRNA.	Biotin probes hybridize to rRNA, removed via magnetic beads.
Advantages	High efficiency, good specificity, works across species.	High specificity, efficient, works across species.	Fast (3 min), simple one-step, no purification, high detection.	Highly efficient, broad species compatibility, high specificity.
Limitations	Requires species-specific probes.	Requires species-specific DNA probes.	Needs specialized kits.	Higher cost.

 

Figure 3. Schematic illustration of rRNA removing

Step 2: mRNA Fragmentation Strategies

After mRNA purification, there are two main strategies for constructing an NGS library:

1)Reverse Transcription First, cDNA Fragmentation Later:

mRNA is first reverse-transcribed using Oligo(dT) primers to generate long cDNA. The cDNA is then converted into double-stranded cDNA (dsDNA) and fragmented using either enzymatic digestion or mechanical shearing. Library preparation then follows the standard DNA library workflow.

2)mRNA Fragmentation First, Reverse Transcription Later:

mRNA is first fragmented using methods such as alkali treatment, metal ion treatment (Mg²⁺ or Zn²⁺), or enzyme digestion (e.g., RNase III). After fragmentation, first-strand cDNA synthesis must be performed immediately to prevent RNA degradation.

For metal ion fragmentation, the conditions can be adjusted based on the desired fragment size:

• 150–200 bp: 94°C, 15 min
• 200–300 bp: 94°C, 10 min
• 250–550 bp: 94°C, 5 min

These two approaches result in different transcript coverage preferences. See the figure below for details.

 Figure 4. Transcript coverage bias of different library preparation methods.

The red line represents RNA fragmentation before reverse transcription, while the green line represents cDNA fragmentation after reverse transcription.

Fragmenting mRNA before reverse transcription mainly generates sequencing reads that evenly cover the gene body. In contrast, reverse transcription using Oligo(dT) primers tends to produce reads biased toward the 3' end of transcripts. Therefore, for mRNA-Seq, it is generally recommended to fragment mRNA first and then perform reverse transcription to achieve more uniform transcript coverage.

Step 3: Synthesis of Double-Stranded cDNA

In mRNA library construction, there are two main types of libraries: standard (non-strand-specific) and strand-specific libraries. The key difference lies in the nucleotides used during second-strand cDNA synthesis.

• For standard libraries, regular dNTPs are used.
• For strand-specific libraries, dUTP replaces dTTP in the dNTP mix during second-strand synthesis. This incorporates uracil into the second strand.

Later, the strand containing uracil can be selectively removed, enabling strand specificity. This is typically done by:

• Using enzymes that block amplification of the uracil-containing strand, or
• Treating with Uracil-DNA Glycosylase (UDG) to degrade the uracil-containing strand.

Important Tips for Successful mRNA Library Preparation

Here are some handy tips to help you avoid common pitfalls during mRNA library prep:

Tip 1: mRNA library prep requires high RNA integrity. Only mostly intact mRNA can produce reliable, high-quality libraries.

Tip 2: When using poly(A) magnetic bead purification, remember that the beads capture sequences near the 3' poly(A) tail. If mRNA is degraded, fragments containing the 5' end won’t be captured and are lost during washing (see Figure 8). This leads to biased data favoring the 3' end.

Tip 3: To ensure you sequence as complete mRNA molecules as possible, perform RNA quality control before library prep.
Use instruments like the Agilent Bioanalyzer 2100 to assess RNA integrity by analyzing 18S and 28S rRNA peaks. Sharp, high peaks indicate less degradation and higher integrity.
The instrument reports an RNA Integrity Number (RIN) from 0 (degraded) to 10 (intact). For best results, use RNA samples with RIN ≥ 8.0.

Product List

Category		Name	Cat. No.
RNA Lib Prep	Dual-mode(Strand specific & Non Strand specific)	Hieff NGS™ Ultima Dual-mode RNA Library Prep Kit	12308ES
	Premix version	Hieff NGS™ EvoMax RNA Library Prep Kit (Strand-specific)	12340ES
		Hieff NGS™ EvoMax RNA Library Prep Kit（dNTP）（Premix and Sealing Version）	12341ES
mRNA isolation	Eukaryotic mRNA	Hieff NGS™ mRNA Isolation Master Kit V2	12629ES
rRNA depletion	Human/Mouse/Rat	Hieff NGS™ MaxUp Human rRNA Depletion Kit ( Human/Mouse/Rat)	12257ES
	Human/Mouse/Rat	Hieff NGS™ MaxUp rRNA Depletion Kit (Plant)	12254ES
		Hieff NGS™ RNA Cleaner	12602ES