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November 2022 | Genomics

Top 3 tips for low-input RNA-seq success

Mapping the route from 500 pg RNA to robust RNA-seq data

Are you wrestling with limited RNA amounts for your gene expression or whole transcriptome studies? Low-input RNA library prep can be immensely challenging. If you’re working with precious yet low-quality, fragmented or FFPE RNA samples, mRNA enrichment and ribosomal RNA (rRNA) removal steps can cause further loss of RNA before the cDNA synthesis steps. Multiple enzymatic and bead cleanup steps after cDNA synthesis can result in a loss of diversity during each step. rRNA contamination can reduce on-target reads, making RNA-seq more cost- and resource-intensive.

So how do you overcome such obstacles that get in the way of your RNA-seq analysis insights?

Here are our top 3 tips:

1. Implement an effective rRNA removal method

If you’re spending hours optimizing RNA-seq and struggling to achieve consistent, on-target reads, rRNA could be to blame. Due to its sheer abundance but low scientific value, rRNA can potentially jeopardize your RNA-seq workflow, compromising your ability to detect low-abundance RNA transcripts of interest. The effects of rRNA contamination become especially pronounced with low-input RNA-seq. 

Efficient rRNA removal is, therefore, a critical but highly overlooked step in RNA-seq optimization. Employing a reliable rRNA removal strategy before low-input RNA-seq library prep ensures you don’t waste your read budget on rRNAs. Ultimately, this means saving on sequencing costs and resources and getting the most out of your low-input RNA sample. 

Our QIAseq FastSelect rRNA removal technology is a game changer for your low-input RNA-seq workflow. It is dramatically faster and easier than alternative rRNA removal methods, which take several hours, require multiple steps, work solely on unfragmented RNA or simply don’t remove enough rRNA. QIAseq FastSelect removes >95% rRNA/globin mRNA in a single step. All it takes is 14 min (or <1 hour, depending on the organism). With QIAseq FastSelect, you achieve exceptional results even with FFPE and fragmented RNA samples. Learn about QIAseq FastSelect here

2. Choose library prep chemistry optimized for use with minimal RNA amounts

When you’ve only got limited amounts of sample, you don’t have room for maneuvering. This means less trial and error when it comes to the library prep chemistry you choose to invest in. With such challenges in mind, we’ve recently developed the QIAseq UPXome RNA Library Kit – a low-input library prep technology designed to work with as little as 500 pg RNA. Whether you’re interested in 3’ RNA-seq or complete transcriptome RNA-seq, you can do both with this highly flexible and versatile kit. Integrated QIAseq FastSelect rRNA removal technology ensures you aren't making unwanted ribosomal cDNA. By removing >95% of unwanted rRNA, you can increase on-target reads, even with low RIN value samples. Learn about UPXome technology here.

3. Adopt a streamlined workflow with fewer steps

Whether it is rRNA removal, 3’ RNA-seq or complete transcriptome RNA-seq library prep, the more complex the workflow, the greater the scope for error and sample loss. Workflow complexity and the number of steps – especially those that involve manual handling – are even more of a consideration when battling against sample loss in low-input RNA-seq.

With our QIAseq FastSelect rRNA removal technology, you can dramatically cut down the number of pipetting steps, protecting limited RNA samples from further loss. 


What if you could go from RNA sample to RNA-seq data in just 6 hours? This can be your RNA-seq reality. Our new QIAseq UPXome RNA Library Kits provide a fast and efficient 6-hour protocol that’s easily automatable. Automation capabilities mean you can achieve greater standardization, reduce handling errors and save time when working with challenging low-input RNA library prep samples, whether for 3’ RNA-seq or complete transcriptome RNA-seq.

Re-imagining the potential of RNA-seq

What kinds of new biological questions could you answer if you could generate high-quality RNA-seq data from 1 ng or less of RNA? Unlock the mysteries of the transcriptome with dedicated technologies designed to help you succeed in your research quest.

Need further resources to help you combat low-input RNA-seq challenges?

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Watch the on-demand webinar

Title: How to resolve challenges with low-input RNA-seq library prep – maximizing whole transcriptome sequencing and analysis using a flexible and customizable strategy
Speaker: Samuel Rulli, Ph.D., Director, Global Product Management, RNA-seq profiling, NGS assay technologies, QIAGEN