Best practices for fecal handling and storage before nucleic acid isolation

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 QIAGEN’s RNAprotect Tissue Reagent or Thermo Fisher’s RNAlater™ are also effectively stable. When using the RNAprotect Tissue Reagent, QIAGEN scientists recommend using our supplementary protocol for best results.
   
   
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 DNA, 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! 

[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.