Best practices for fecal handling and storage before nucleic acid isolation

MicrobiomeJune2019

Getting biodiversity data from fecal samples is tricky. How materials are handled and stored can
mean the difference between accurately pinpointing diverse and delicate gut flora and getting
woefully unclear sequencing data. Following the previous post on the subject, we received
interest in learning more about how researchers can get more quality data from even small
amounts of fecal material.

1) Storage is of course, still key. Freeze and thaw cycles are harmful to delicate microbial
cell walls and can also cause degeneration of RNA material but improve collection of
DNA among some species of microbes [1]. Snap freezing can minimize potential
damage and is useful for samples being kept for long-term storage, although repeated
freeze-thaw cycles will still degrade DNA [2]. The best way to store fecal material is at
room temperature or on cold packs and use within 14 days [2], but materials stored with
Thermo Fisher’s RNAlater™ are also effectively stable. QIAGEN scientists, however,
recommend only using 100 mg of sample when using RNAlater to avoid coating of silica
membranes, as this results in poor nuclear material recovery.

2) Stabilizers such as betamercaptoethanol (BME) or phenol:chloroform:isoamyl, pH 7-8
(PCl) can also be effective, if used in the lysis buffer stage. However, there may be
drawbacks: BME can permanently destroy the RNases you may be targeting. PCl needs
to be used quickly on frozen samples to protect material from further degradation. Speed
is key for stabilization, especially if you’re working with delicate materials and performing
longer protocols.

3) Eliminating sample impurities and inhibitors: Among the biggest difficulties to
getting accurate data is in separating the wheat from the chaff. In fecal microbiome
studies, that chaff includes impurities like undigested food and other non-microbiome biological materials. While QIAGEN offers powerful kits like our QIAamp® PowerFecal®
Pro, DNeasy®, RNeasy®, MagAttract® and AllPrep® fecal kits (Table 1), to help remove
heme and other materials that inhibit downstream analysis, sometimes there is still more
debris that needs to be removed ahead of isolating microbial nucleic acids. A spin
protocol developed at McGill University in Montreal effectively does just that [3]. This
protocol takes a small 0.2 g amount of sample material, spins it in saline with a filter for
15 minutes to remove solids before taking the remaining bacterial pellet through the kit
protocol steps.

4) Dead cells can also be an issue. Our scientists recommend a spin filter purification
using 70% ethanol during a binding step to help improve the capture of larger molecular
weight RNAs, but for low molecular weight RNA or microRNAs, continue to use 100%
ethanol when spinning.

Every lab faces their own challenges in obtaining unbiased microbiome data from difficult
samples. While we have a host of webinars on the subject, we also would like to hear from you
about what works for you and your lab. Feel free to email us or drop us a message in the
comments section below!

Table 1

Kit Nucleic acid Automated on High throughput Cat #
QIAamp PowerFecal Pro DNA Kit DNA QIAcube 51804
DNeasy 96 PowerSoil Pro QIAcube HT Kit DNA QIAcube HT 47021
QIAamp 96 PowerFecal
QIAcube HT Kit*
DNA QIAcube HT 51531
RNeasy PowerMicrobiome® Kit RNA QIAcube 26000-50
AllPrep PowerViral® DNA/RNA Kit DNA, RNA QIAcube 28000-50
AllPrep PowerFecal DNA/RNA Kit DNA, RNA QIAcube 80244
MagAttract PowerMicrobiome DNA/RNA Kits DNA, RNA KingFisher™ epMotion® 27500-4-EP 27600-4-KF


References

[1] Fouhy F, Deane J, Rea MC et al. The effects of freezing on faecal microbiota as determined
using miseq sequencing and culture-based investigations. PLoS ONE 10(3), e0119355 (2015).
[2] Thomas V, Clark J, Doré J. Fecal microbiota analysis: an overview of sample collection
methods and sequencing strategies. Future Microbiology 10(9) (2015)
[3] Vincent C, Miller MA, Manges AR, Dewar K. Quantification of bacterial survival in fecal
filtrates after a freeze/thaw cycle. Keystone Symposia, Big Sky, MT, April 1-6 2014.

Heather Martinez

Associate Director, Global Strategic Marketing, Microbiome

Heather Martinez received her PhD in Biomedical Sciences from the University of California, San Diego in 2007 and has a scientific background in subjects including microbiome research, molecular biology, cell biology and protein biochemistry. She was Associate Director of Marketing at MO BIO Laboratories, Inc. which was acquired by QIAGEN in 2015. Currently, Dr. Martinez is responsible for strategic marketing for QIAGEN’s microbiome portfolio.

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