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Top 5 Tips microRNA NGS in serum/plasma

MicroRNAs are stable in liquid biopsies and hold great promise as minimally invasive biomarkers for a wide range of diseases. However, biofluids are challenging as they contain low levels of RNA, high levels of inhibitors and are susceptible to many pre-analytical variables. Performing microRNA Next Generation Sequencing (NGS) in biofluids requires experience and specialized protocols.

Our pioneering work with biofluids has enabled our Expert Services to develop sample handling recommendations and optimized protocols for NGS in biofluids to ensure reliable results.

1. Design the optimal experiment

  • Include sufficient biological replicates per group. A recent publication recommends minimum 6 biological replicates per group (Schurch et al., 2016). We recommend prioritizing biological replicates (samples from different individuals) over technical replicates (sequencing the same sample multiple times).
  • Consider a pilot study or power analysis to determine the number of biological replicates needed to reach statistical significance in your sample set. A power analysis is always provided with data from Exiqon Services.
  • Don’t pool biofluid samples from different individuals – the results from pools are not borne out by individual samples, and variation between individuals cannot be assessed if the samples are pooled (Rajkumar et al., 2015)
  • Combine NGS with qPCR to increase the number of samples you can include in your study. By performing an initial screening using NGS, and selecting candidate microRNAs for profiling using custom PCR panels, a much larger number of samples can be profiled, thereby increasing statistical confidence in the results (Figure 1). This approach is also much more cost-effective than profiling the same number of samples using NGS alone.
Use NGS and PCR Panels to increase the number of samples in a microRNA profiling study

Figure 1: Use NGS and PCR Panels to increase the number of samples in a microRNA profiling study, thereby increasing statistical confidence in the results. Exiqon Services can perform NGS and qPCR profiling (using the miRCURY LNA™ Universal RT microRNA PCR System) tailored to your study.

2. Use best practice serum/plasma sample collection

  • Use a well-described standardized procedure for sample collection. Try to ensure all samples are handled in the same way, and sample collection procedures are consistent between different collection sites. Read more and download the EDRN standard operating procedures.
  • Use good quality collection tubes, e.g., BD Vacutainer. Ensure all samples are collected using the same tube type, to minimize technical variation.
  • Avoid heparin tubes. Heparin inhibits downstream enzymatic processes.
  • Process whole blood into serum or plasma as soon as possible. If blood cannot be processed immediately, it should be stored for maximum 4 hours at room temperature (storage at 4 °C may result in lysis of thrombocytes and contamination of the cell-free microRNA profile).
  • Store serum/plasma samples at -80 °C in RNase-free tubes. Avoid multiple freeze-thaw cycles.

3. Avoid biased results due to hemolysis or cellular contamination

  • Use good quality collection tubes (e.g., BD Vacutainer) to minimize hemolysis which can affect the cell-free microRNA profile.
  • Spin to remove cells – before freezing the samples. Spin serum/plasma for 5 minutes at 3,000 x g to remove cells prior to freezing. This will avoid contamination of the cell-free microRNA profile.
  • Check for signs of hemolysis. Only severely hemolyzed samples will appear visibly red. We have developed a qPCR based hemolysis indicator (that measures levels of two endogenous microRNAs, Blondal et al., 2013), which has been demonstrated to be the most sensitive method to detect hemolysis (Shah et al., 2016). Hemolysis QC for biofluid samples is performed as standard by Exiqon Services.

4. Use QC procedures optimized for liquid biopsies

  • Standard RNA QC is not suitable for biofluids – the low levels of RNA in biofluids are below the reliable range of standard methods (e.g. NanoDrop, Qubit and Bioanalyzer).
  • Use qPCR-based methods for QC of biofluid RNA. We have developed qPCR QC for biofluids, which uses a combination of RNA spike-ins and endogenous microRNAs to monitor RNA isolation efficiency, inhibitors, hemolysis and detect outliers. qPCR QC for biofluid samples is performed as standard by Exiqon Services using the ExiSEQ NGS sample QC kit – SmallRNA/microRNA.
  • Perform Sequencing QC. As part of the QC using the ExiSEQ NGS sample QC kit – SmallRNA/microRNA, Exiqon Services add the 52 RNA spike-ins during the RNA isolation step, which are used to monitor the linearity and reproducibility of Biofluids microRNA sequencing (Figure 2).
Excellent technical reproducibility of microRNA sequencing from plasma (replicate RNA isolations) by Exiqon Services

Figure 2: Excellent technical reproducibility of microRNA sequencing from plasma (replicate RNA isolations) performed by Exiqon Services. Reliable microRNA NGS results can be obtained from biofluids, provided protocols optimized for biofluids are used at every step, from sample collection to data analysis.

5. Ensure reads are reliable – and validate your results

  • Use the RNA isolation method optimized for biofluids NGS. We have tested a wide range of different RNA isolation methods, and found that no commercially available kit performed well enough. So, we developed our own optimized RNA isolation method specifically for NGS biofluids, which is unique to Exiqon Services.
  • Do not use a carrier RNA when isolating RNA from biofluids intended for NGS.
  • Library preparation must be optimized for low content samples. Exiqon Services have developed a library preparation method optimized for the low concentration of starting material found in biofluids.
  • Ensure sequencing depth is sufficient for biofluids. Exiqon Services take great care to ensure that the sequencing depth is sufficient to allow accurate analysis of even the lowly expressed microRNAs found in biofluids.
  • Map microRNA reads to relevant databases. Exiqon Services use a specialized automated microRNA data analysis pipeline that maps reads to the latest version of miRBase as well as the appropriate reference genome, and also identifies novel microRNAs and isomiRs.
  • Understand how reliable a read is. It is a good idea to normalize to read number (TPM, or Tags per Million mapped reads) and set a TPM threshold so you can focus on the most reliable microRNA reads. Exiqon Services performs the NGS data analysis as standard, and will discuss with you the most appropriate threshold for your study.
  • Remember read number is not directly related to abundance. NGS involves multiple ligation and PCR reactions, hence read numbers are not quantitative – results should be normalized and used to identify relative differences between samples.
  • Always validate your NGS results using another technique, e.g., qPCR. NGS reads with low TPM or small-fold changes between groups should be carefully validated. Exiqon Services perform qPCR validation and offer assistance with selection of appropriate reference genes for normalization.

Additional resources

Exiqon Services
Complete sample-to-answer microRNA profiling and validation services.
Learn more

Biofluids Guidelines
The comprehensive guide to analyzing microRNAs in liquid biopsies by qPCR or NGS.
Download guidelines

See how biofluid microRNA profiles compare by NGS and qPCR.
Download TechNote

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Biofluids microRNA Sequencing

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