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Extraction of DNA from Murine Fecal Pellets for Downstream Phylogenetic Microbiota Analysis by Next-generation Sequencing

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Aug 2017



Mouse models are widely used to evaluate the potential impact of the gut microbial composition on health and disease. Standardized protocols for sampling and storing murine feces, as well as for extracting DNA from these fecal pellets are needed to limit experimental variation between different studies. Both efficient lysis of the microbiota and the quality of the obtained fecal DNA are important for allowing the downstream next-generation sequencing to cover the phylogenetic diversity of both Gram-negative and Gram-positive bacteria living in the mouse gut. Here we present a detailed protocol for fecal sample collection and DNA extraction that we validated in a study on the impact of inflammasomes on the murine gut microbiota. This protocol for DNA extraction from murine fecal pellets utilizes a combination of mechanical and chemical lysis, which aligns with the procedure that was recently recommended as a benchmark protocol for DNA extraction from human feces.

Keywords: Gut microbiota (肠道微生物群), Fecal DNA (粪便DNA), Mouse feces (小鼠粪便), DNA extraction (DNA提取), 16S phylogenetic analysis (16S系统发生分析), Next-generation sequencing (新一代测序)


Limiting technical variation within as well as between laboratories is imperative for reproducibility and hence for scientific progress from experimental research. Within the expanding gut microbiota research community, a plethora of methodologies are used to profile the phylogenetic composition of the intestinal ecosystem. Each step in this microbiota analysis process is subject to technical variation depending on the protocol or the materials used. For instance, comparing several protocols to extract DNA from murine feces showed striking differences in the obtained results even within the same laboratory (Ferrand et al., 2014). Therefore, it is clear that standardized protocols are needed to enable meta-analyses of multiple different studies.

For analyzing the human fecal microbiota, a large international consortium of researchers recently compared the effects of numerous technical approaches in every single step of the gut microbiota analysis pipeline in several independent laboratories (Costea et al., 2017). This study identified differences in the DNA extraction method as the biggest influence on the downstream gut microbiota analysis results. Based on the obtained DNA quality as well as on the reproducibility between different laboratories, the so-called ‘Protocol Q’ was identified as the best one and was proposed as a benchmark for extracting DNA from human feces (Costea et al., 2017).

Although such multi-centered comparative studies have not been performed for murine fecal DNA extraction protocols, we recently reported a gut microbiota profiling study in mice using a protocol similar to the Protocol Q recommended for human fecal DNA extraction (Mamantopoulos et al., 2017). Like the latter, our protocol uses a combination of mechanical bead-beating and chemical lysis with the QIAGEN QiaAmp® Stool Kit. Indeed, it has been reported that mere chemical lysis of feces results in an underrepresentation of DNA from Gram-positive bacteria that have a thicker cell wall (Salonen et al., 2010). In contrast, both Protocol Q and our protocol detailed below are expected to result in efficient lysis of both Gram-positive and Gram-negative bacteria.

Materials and Reagents

  1. Filter tip, clear, sterile F. Gilson P1000, 60 PCS/Box (Greiner Bio One International, catalog number: 740288 )
  2. Filter tip, clear, sterile F. Gilson P-200, 96 PCS/Box (Greiner Bio One International, catalog number: 739288 )
  3. Standard filter tip, 20 µl, clear, universal, sterile, 96 pieces per rack (Greiner Bio One International, catalog number: 774288 )
  4. Soil grinding SK38 2 ml tubes (Bertin Technologies, catalog number: KT03961-1-006.2 )
  5. Eppendorf® Tubes 3810X, 1.5 ml, g-safe® centrifugation stability, Eppendorf QualityTM, colorless, 1,000 pcs. (Eppendorf, catalog number: 0030125150 )
  6. Ethanol absolute, EMSURE® ACS, ISO, Reag. Ph. Eur. analytical reagent (Merck, MilliporeSigma, catalog number: 1.00983.1000 )
  7. UltraPureTM DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, GibcoTM, catalog number: 10977035 )
  8. QIAamp® Fast DNA Stool Mini Kit (QIAGEN, catalog number: 51604 ), containing the following:
    1. QIAamp Mini Spin Columns
    2. Collection Tubes (2 ml)
    3. InhibitEX® Buffer
    4. Proteinase K
    5. Buffer AL
    6. Buffer AW1 concentrate
    7. Buffer AW2 concentrate
    8. Buffer ATE


  1. Finnpipette F1, 100 to 1,000 μl (Thermo Fisher Scientific, catalog number: 4641100N )
  2. Finnpipette F1, 20 to 200 μl (Thermo Fisher Scientific, catalog number: 4641080N )
  3. Finnpipette F1, 2 to 20 μl (Thermo Fisher Scientific, catalog number: 4641060N )
  4. Beakers
  5. -80 °C freezer
  6. Precellys®24 (Bertin Technologies, catalog number: EQ03119.200.RD00.0 )
  7. Thermoshaker with heating block for 24 x1.5 ml microtubes (Grant Instruments, catalog number: PHMT-PSC24N )
  8. Microcentrifuge 5417R with rotor for 1.5/2 ml tubes (Eppendorf, model: 5417 R , catalog number: 22 62 180-7)
  9. Vortex mixer (Merck Eurolab, catalog number: MELB 1719 )
  10. NanoDrop spectrophotometer


  1. Fecal sample collection
    1. Collect 1-2 fresh fecal pellet(s) from mice into a soil grinding SK38 2 ml tube. Fresh fecal pellets are collected by holding the mouse in one hand, during which the mouse can defecate directly in an SK38 2 ml tube held in the other hand. Alternatively, individual mice can be put in sterile beakers for a couple of minutes, after which fresh fecal pellets can be collected from the beakers. It is important to collect fecal samples from all animals within the experimental cohort at the same time period of day, since it was reported that the gut microbiota composition displays diurnal oscillations (Thaiss et al., 2014).
    2. Store the soil grinding SK38 2 ml tubes containing the fecal pellet(s) at -80 °C until further processing. Fecal DNA extraction can also be initiated immediately from fresh fecal pellets, but all samples within an experimental cohort should be either all fresh or all frozen to avoid storage artifacts, as freezing fecal samples was shown to influence the ratio of Firmicutes to Bacteroidetes (Bahl et al., 2012).

  2. Fecal DNA extraction
    Fecal DNA extraction is performed using the QIAamp® Fast DNA Stool Mini Kit according to the manufacturer’s instructions with additional mechanical lysis by bead-beating as detailed below:
    1. Add 1 ml InhibitEX® buffer to the soil grinding SK38 2 ml tubes containing fecal pellet(s). Fecal pellets taken from the -80 °C freezer can be used immediately, as equilibrating to room temperature is not required.
    2. Homogenize fecal pellets in 1 ml InhibitEX® buffer by bead-beating using the Precellys®24 for 2 x 30 sec at 6,500 rpm.
    3. Transfer the fecal homogenate from Step B2 to sterile 1.5 ml microcentrifuge tubes, taking care to avoid transferring the zirconium beads.
    4. Heat the fecal homogenate in a thermoshaker for 5 min at 70 °C while shaking at 1,100 rpm in order to promote further lysis of the bacteria.
    5. Centrifuge the fecal homogenate from Step B4 at 20,800 x g for 1 min at room temperature in order to pellet the stool particles.
    6. In the meantime, pipet 15 µl Proteinase K from the QIAamp® Fast DNA Stool Mini Kit to fresh sterile 1.5 ml microcentrifuge tubes.
    7. Pipet 200 µl fecal homogenate supernatant from Step B5 into the Proteinase K containing 1.5 ml microcentrifuge tubes.
    8. Add 200 µl buffer AL to the 1.5 ml microcentrifuge tubes containing 200 µl supernatant and Proteinase K (from Step B7) and vortex for 15 sec to form a homogeneous suspension.
    9. Heat the suspension in the thermoshaker for 10 min at 70 °C while shaking at 1,100 rpm.
    10. Add 200 µl absolute ethanol to the suspension, and vortex to mix.
    11. Transfer 600 µl of the resulting lysate carefully onto the QIAamp spin columns provided in the QIAamp® Fast DNA Stool Mini Kit.
    12. Close the QIAamp spin column and centrifuge at room temperature for 1 min at 20,800 x g.
    13. Place the QIAamp spin column in a new 2 ml collection tube and discard the old collection tube containing the filtrate.
    14. Add 500 µl buffer AW1 to the QIAamp spin column.
      Note: Buffer AW1 is provided as a concentrate. When using a fresh bottle, first add 25 ml absolute ethanol to the AW1 concentrate and mix thoroughly by resuspending.
    15. Close the QIAamp spin column and centrifuge for 1 min at room temperature at 20,800 x g.
    16. Place the QIAamp spin column in a new 2 ml collection tube and discard the old collection tube containing the filtrate.
    17. Add 500 µl buffer AW2 to the QIAamp spin column.
      Note: Buffer AW2 is provided as a concentrate. When using a fresh bottle, first add 30 ml absolute ethanol to the AW2 concentrate and mix thoroughly by resuspending.
    18. Close the QIAamp spin column and centrifuge for 3 min at room temperature at 20,800 x g.
    19. Place the QIAamp spin column in a new 2 ml collection tube and discard the old collection tube containing the filtrate.
    20. Centrifuge again for 3 min at room temperature at 20,800 x g in order to reduce the chances of buffer AW2 carryover.
    21. Place the QIAamp spin column in a fresh 1.5 ml microcentrifuge tube and discard the old collection tube containing the filtrate.
    22. Add 200 µl UltraPureTM DNase/RNase-Free Distilled Water to the QIAamp spin column to elute the DNA.
      Note: DNA can also be eluted using 200 µl of the buffer ATE provided by the QIAamp® Fast DNA Stool Mini Kit.
    23. Incubate for 1 min at room temperature.
    24. Centrifuge at room temperature for 1 min at 20,800 x g to collect the DNA.
    25. Discard the QIAamp spin columns.
    26. Close the 1.5 ml microcentrifuge tubes containing the eluate and store the DNA at -20 °C until further use. The amount of fecal DNA obtained with this protocol varies per sample, and depends on the amount of stool used to extract DNA. In our hands, the amount of fecal DNA obtained varies between 50 and 500 ng DNA per mg stool, with an average of about 150 ng DNA per mg stool. Since one fecal pellet weighs at least 20 mg, one can expect to obtain at least 1 μg of fecal DNA using this protocol. Thus, as the fecal DNA is dissolved in 200 μl of UltraPureTM DNase/RNase-Free Distilled Water (see Step A22), the obtained concentration of fecal DNA from any given sample will be at least 5 ng/μl.

Data analysis

Concentration and quality of the obtained fecal DNA can be measured by NanoDrop analysis, in which the absorbance ratios at 260 nm/230 nm and 260 nm/280 nm can be determined to evaluate the purity of the extracted DNA, which should be around 2 and 1.8, respectively. Downstream phylogenetic 16S rDNA microbiota analysis starts with a PCR on 25 ng of fecal DNA. Therefore, since this protocol generates at least 1 μg of DNA at a concentration of 5 ng/μl, the quantity of obtained DNA is not a limiting factor. Further procedures for phylogenetic 16S rDNA microbiota analysis are outlined in our previous study (Mamantopoulos et al., 2017).


A.W. is supported by the Odysseus grant G.0C49.13N from the Fund for Scientific Research-Flanders, and is a post-doctoral fellow with the Fund for Scientific Research-Flanders. The authors do not have any conflicts of interest.


  1. Bahl, M. I., Bergstrom, A. and Licht, T. R. (2012). Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis. FEMS Microbiol Lett 329(2): 193-197.
  2. Costea, P. I., Zeller, G., Sunagawa, S., Pelletier, E., Alberti, A., Levenez, F., Tramontano, M., Driessen, M., Hercog, R., Jung, F. E., Kultima, J. R., Hayward, M. R., Coelho, L. P., Allen-Vercoe, E., Bertrand, L., Blaut, M., Brown, J. R. M., Carton, T., Cools-Portier, S., Daigneault, M., Derrien, M., Druesne, A., de Vos, W. M., Finlay, B. B., Flint, H. J., Guarner, F., Hattori, M., Heilig, H., Luna, R. A., van Hylckama Vlieg, J., Junick, J., Klymiuk, I., Langella, P., Le Chatelier, E., Mai, V., Manichanh, C., Martin, J. C., Mery, C., Morita, H., O'Toole, P. W., Orvain, C., Patil, K. R., Penders, J., Persson, S., Pons, N., Popova, M., Salonen, A., Saulnier, D., Scott, K. P., Singh, B., Slezak, K., Veiga, P., Versalovic, J., Zhao, L., Zoetendal, E. G., Ehrlich, S. D., Dore, J. and Bork, P. (2017). Towards standards for human fecal sample processing in metagenomic studies. Nat Biotechnol 35(11): 1069-1076.
  3. Ferrand, J., Patron, K., Legrand-Frossi, C., Frippiat, J. P., Merlin, C., Alauzet, C. and Lozniewski, A. (2014). Comparison of seven methods for extraction of bacterial DNA from fecal and cecal samples of mice. J Microbiol Methods 105: 180-185.
  4. Mamantopoulos, M., Ronchi, F., Van Hauwermeiren, F., Vieira-Silva, S., Yilmaz, B., Martens, L., Saeys, Y., Drexler, S. K., Yazdi, A. S., Raes, J., Lamkanfi, M., McCoy, K. D. and Wullaert, A. (2017). Nlrp6- and ASC-dependent inflammasomes do not shape the commensal gut microbiota composition. Immunity 47(2): 339-348 e334.
  5. Salonen, A., Nikkila, J., Jalanka-Tuovinen, J., Immonen, O., Rajilic-Stojanovic, M., Kekkonen, R. A., Palva, A. and de Vos, W. M. (2010). Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. J Microbiol Methods 81(2): 127-134.
  6. Thaiss, C. A., Zeevi, D., Levy, M., Zilberman-Schapira, G., Suez, J., Tengeler, A. C., Abramson, L., Katz, M. N., Korem, T., Zmora, N., Kuperman, Y., Biton, I., Gilad, S., Harmelin, A., Shapiro, H., Halpern, Z., Segal, E. and Elinav, E. (2014). Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis. Cell 159(3): 514-529.


小鼠模型被广泛用于评估良好的微生物组成对健康和疾病的潜在影响。 为了限制不同研究之间的实验差异,需要用于取样和储存鼠粪的标准化方案,以及从这些粪便颗粒中提取DNA。 微生物群的有效裂解和产生的粪便DNA对于允许下游的下一代测序覆盖革兰氏阴性菌和革兰氏阳性菌的系统发育多样性是重要的。 在这里,我们提出了一个详细的粪便样本采集和DNA提取方案,我们在炎症小鼠对小鼠肠道微生物群的影响的研究中进行了验证。 这种从鼠粪球中提取DNA的方案利用了机械和化学溶解的组合,这与最近推荐的用于从人类粪便中提取DNA的基准方案一致。


为了分析人类粪便微生物研究人员组成的大型国际财团日前相比,在几个独立的实验室以及微生物分析流水线的每一个步骤的许多技术方法的效果(科斯泰亚的等的,2017年) ,这项研究确定了DNA提取方法的差异,作为对下游微生物群分析结果的最大影响。基于所获得的DNA的质量,以及在不同的实验室之间的再现性,即所谓的“协议Q”什么确定为最好的一个,什么建议作为用于提取从人类粪便DNA的基准(科斯泰亚等。 ,2017)。

虽然寻求多中心比较研究尚未执行用于鼠粪便DNA提取方案,我们最近报道在小鼠中良好的微生物群分析研究使用类似于推荐用于人类粪便DNA提取的协议Q上协议(Mamantopoulos 等。 ,2017)。像后者,我们的协议使用机械珠磨和化学裂解与QIAGEN QIAamp试剂®凳子试剂盒的组合。事实上,已报道在没有粪便结果单纯化学裂解从革兰氏阳性细菌中的DNA的代表性不足thathave较厚细胞壁(Salonen的等人,2010)。相比之下,协议Q和我们的协议预计会导致革兰氏阳性菌和革兰氏阴性菌的有效裂解。

关键字:肠道微生物群, 粪便DNA, 小鼠粪便, DNA提取, 16S系统发生分析, 新一代测序


  1. 过滤器尖端,透明,无菌F. Gilson P1000,60盒/箱(Greiner Bio One International,目录号:740288)
  2. Gilson P-200,96 PCS /盒(Greiner Bio One International,目录号:739288)。
  3. 标准过滤器尖端,20微升,透明,通用,无菌,每个机架96件(Greiner Bio One International,目录号:774288)。
  4. 土壤研磨SK38 2毫升管(Bertin Technologies,目录号:KT03961-1-006.2)。
  5. 的Eppendorf ®管3810X,将1.5ml,G-安全®离心稳定性,Eppendorf优质 TM ,无色,1000个。 (Eppendorf,目录号:0030125150)
  6. 无水乙醇,EMSURE ACS,ISO,Reag。 Ph.Eur。分析试剂(Merck,Millipore Sigma,目录号:1.00983.1000)
  7. UltraPure TM DNase / RNase-Free蒸馏水(Thermo Fisher Scientific,Gibco TM,目录号:10977035)。
  8. QIAamp Fast DNA Stool Mini试剂盒(QIAGEN,目录号:51604),包含以下内容:
    1. QIAamp迷你旋转柱
    2. 收集管(2毫升)
    3. InhibitEX Buffer
    4. 蛋白酶K
    5. 缓冲区AL
    6. 缓冲AW1集中
    7. 缓冲AW2浓缩液
    8. 缓冲区ATE


  1. Finnpipette F1,100至1,000μl(Thermo Fisher Scientific,目录号:4641100N)。
  2. Finnpipette F1,20至200μl(Thermo Fisher Scientific,目录号:4641080N)
  3. Finnpipette F1,2至20μl(Thermo Fisher Scientific,目录号:4641060N)。
  4. 烧杯
  5. -80°C冷冻机
  6. Precellys 24(Bertin Technologies,目录号:EQ03119.200.RD00.0)
  7. 带有加热块的Thermoshaker用于24 x1.5 ml微管(Grant Instruments,目录号:PHMT-PSC24N)
  8. 具有用于1.5 / 2ml管的转子的微量离心机5417R(Eppendorf,型号:5417R,目录号:2262 180-7)。
  9. 涡旋混合器(Merck Eurolab,目录号:MELB 1719)
  10. NanoDrop分光光度计


  1. 粪便样品收集
    1. 从小鼠中收集1-2个新鲜的粪便颗粒到土壤研磨的SK38 2ml试管中。一只手握住老鼠收集新鲜的粪便颗粒,在此过程中,可以直接将粪便排入另一只手握住的SK38 2ml试管中。或者,可将个别小鼠放入无菌烧杯中几分钟,然后从烧杯中收集新鲜的粪粒。重要的是要在一天中在Sametime周期实验队列内收集从所有动物粪便样品是重要的,因为它其中报告DASS模具以及微生物群组成显示昼夜振荡(Thaiss 等人,2014)。
    2. 将含有粪便颗粒的土壤研磨SK38 2ml试管储存在-80℃直至进一步处理。粪便DNA提取,因此可以从新鲜的粪便颗粒立即开始,但内实验队列所有样品应该是所有新鲜或全部冻结,以避免存储的工件,如冷冻其中显示影响厚壁菌门的<比率/粪便样品 to Bacteroidetes (Bahl et。,2012)。

  2. 粪便DNA提取
    粪便DNA提取用QIAamp 执行的®快速DNA粪便Mini试剂盒gemäß制造商的与由珠磨额外的机械裂解指令详述如下:
    1. 加入1ml InhibitEX缓冲液到含有粪便颗粒的土壤研磨SK382ml管中。取自-80°C冰柜的粪便颗粒可以立即使用,因为不需要。
    2. 均质粪便颗粒在1ml Inhibitex ®缓冲器通过使用Precellys珠磨® 24在6500转2×30秒。
    3. 将步骤B2的粪便匀浆转移到无菌的1.5ml微量离心管中,注意避免转移锆珠。
    4. 在70℃加热5分钟的热匀浆中的粪便匀浆,同时在1100rpm振荡,以促进细菌的进一步裂解。
    5. 将来自步骤B4的粪便匀浆在室温下在20,800×gg下离心1分钟以沉淀粪便颗粒。
    6. 在平均时间,移液管15微升蛋白酶K从QIAamp试剂®快速DNA粪便Mini试剂盒到新鲜无菌1.5ml微量管中。
    7. 吸取步骤B5的200μL粪便匀浆上清液到含有1.5ml微量离心管的蛋白酶K中。
    8. 添加200μl缓冲液AL到1.5ml微量管中含有200微升上清液,蛋白酶K(来自步骤B7),并涡旋15秒,以形成均匀的悬浮液。

    9. 在70℃加热10分钟,同时摇动1,100转

    10. 在悬浮液中加入200μl无水乙醇并涡旋混合
    11. QIAamp Fast DNA Stool Mini Kit中提供的QIAamp离心柱。
    12. 关闭QIAamp离心柱,并在室温下以20,800×g g离心1分钟。
    13. 将QIAamp离心柱置于新的2 ml收集管中,丢弃含有滤液的旧收集管。
    14. 向QIAamp离心柱中加入500μl缓冲液AW1。
    15. 关闭QIAamp离心柱,并在20,800×gg室温下离心1分钟。
    16. 将QIAamp离心柱置于新的2 ml收集管中,丢弃含有滤液的旧收集管。
    17. 向QIAamp离心柱中加入500μl缓冲液AW2。
      注意:Buffer AW2是作为浓缩物提供的。当使用新鲜的瓶子时,首先将30毫升无水乙醇加入到AW2浓缩物中,并通过重悬来彻底混合。
    18. 关闭QIAamp离心柱,并在室温下以20,800×g g离心3分钟。
    19. 将QIAamp离心柱置于新的2 ml收集管中,丢弃含有滤液的旧收集管。
    20. 为了减少缓冲AW2残留的机会,在室温下以20,800xg克再次离心3分钟。
    21. 将QIAamp离心柱置于新鲜的1.5ml微量离心管中,丢弃含有滤液的旧收集管。
    22. 向QIAamp离心柱中加入200μlUltraPure™DNase / RNase-Free蒸馏水以洗脱DNA。
      注:“DNA可以因此使用200微升的缓冲液洗脱ATE通过QIAamp试剂 ® 快速DNA粪便Mini试剂盒提供。 /

    23. 在室温下孵育1分钟

    24. 在20,800×g的室温下离心1分钟以收集DNA。
    25. 丢弃QIAamp旋转栏。
    26. 关闭含有洗出液的1.5ml微量离心管,并将DNA储存在-20℃直至进一步使用。这个协议获得的粪便DNA的数量因样本而异,并且取决于用于提取DNA的粪便的量。在我们的手中,获得的粪便DNA的量在每毫升粪便50-500ng DNA之间变化,平均每mg粪便约150ng DNA。由于一个粪便颗粒重量至少为20毫克,所以使用这个方案可以获得至少1微克的粪便DNA。因此,当粪便DNA在200μl的超纯水 TM DNA酶/ RNA酶的蒸馏水(见步骤A22),粪便DNA的从任何给定的样品所获得的浓度将是至少5纳克的溶解/μl。


将所得到的粪便DNA的浓度和质量可以通过纳米滴分析来测定,其中,在260nm / 230 nm和260nm处/ 280nm的吸光度比可以是确定性的开采评价提取的DNA的纯度,这应该是围绕两个和1.8,分别。下游系统发育16S rDNA微生物群分析始于对25ng粪便DNA的PCR。因此,由于该方案以5ng /μl的浓度产生至少1μg的DNA,所获得的DNA量不是限制因素。进一步的系统发育16S rDNA微生物群分析程序在我们以前的研究中概述(Mamantopoulos等,2017)。


A. W.通过从科学研究基金龙龙的奥德修斯授予G.0C49.13N支持,是一个博士后与科研龙龙基金。作者没有任何利益冲突。


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引用:Eeckhout, E. and Wullaert, A. (2018). Extraction of DNA from Murine Fecal Pellets for Downstream Phylogenetic Microbiota Analysis by Next-generation Sequencing. Bio-protocol 8(3): e2707. DOI: 10.21769/BioProtoc.2707.