RNA sequencing (RNA-Seq) is a powerful method for studying the transcriptome qualitatively and quantitatively; it can be used to identify the full catalog of transcripts, precisely define the structure of genes, and accurately measure gene expression levels. Traditional RNA-Seq approaches typically require at least 10,000 cells or 500 ng of total RNA per sample. For experiments that yield lower amounts of starting material, ultra-low input RNA sequencing may be a viable alternative. It provides bulk expression analysis of samples containing as few as ~100 cells or ~10 pg of RNA.
When working with very limited starting material, high-quality RNA is paramount to obtain good sequencing data. If you’re using a next generation sequencing (NGS) provider, be mindful that sample preparation and shipping can significantly affect RNA quality. Let’s examine common questions that arise when submitting these precious materials for ultra-low input RNA-Seq.
Do I submit cells or extracted RNA? Does this impact RNA quality?
Conventional quality control methods cannot be used to analyze ultra-low input RNA samples prior to library preparation. The limit of detection is ~100 ng for the Qubit® fluorometer and ~25 ng for the TapeStation™ electrophoresis system. For low-input samples, the success of RNA extraction won’t be known until after cDNA synthesis and amplification are performed.
As a result, technical expertise is critical when extracting RNA from minute quantities of starting material. The decision to submit cell pellets or extracted RNA should depend, in part, on your proficiency and success record with RNA extractions. If you’re unsure or concerned about potential quality issues, we recommend submitting cells to a reputable lab for RNA extraction. At Azenta Life Sciences, we have extensive experience in extracting RNA from a wide range of low-input and less-than-ideal samples.
What is the best method for shipping ultra-low input samples?
Once you have chosen WHAT you prefer to submit, cells or purified RNA, the next step is to decide HOW you ship the samples to the sequencing provider. The tubes or plates you use can have a major impact on sample recovery. Because of the limited amount of material, RNA binding to the tube surface can significantly reduce sample recovery during library generation.
Typical PCR plates are made of polypropylene (PP) as it’s chemically inert, resistant to solvents, and well suited for injection molding—enabling production of thin-walled tubes for fast heat transfer during thermal cycling. Unfortunately, nucleic acids have been shown to bind to PP tubes, especially in high ionic conditions, despite the very hydrophobic nature of this material. While this may not impact experiments with ample starting material, with ultra-low input samples, every bit counts.
Low binding microplates from Azenta Life Sciences offer reduced nucleic acid binding without the use of surface coatings that can leach and contaminate samples. They are composed of carefully selected PP polymers with low-binding characteristics. These plates have been tested under a broad temperature range and offer maximum nucleic acid recovery after low temperature storage or shipping, making them ideal for ultra-low input samples. Even if you’re submitting cells, it’s advisable to use low-binding plates as RNA extraction may be performed in the same container. We recommend all plates be well sealed with a high-quality foil seal and shipped overnight on dry ice to minimize sample degradation during transit.
Learn more about our ultra-low binding products and how they enable maximum nucleic acid recovery for sensitive NGS applications.
About the Author
Andrea O'Hara, Ph.D., is a Strategic Technical Specialist at Azenta Life Sciences and has over 11 years of experience in next generation sequencing. She earned her Ph.D. from the University of North Carolina at Chapel Hill in genetics and molecular biology.