Materials and Reagents

For gDNA isolation
1. Filter MAXIPENSE^TM, Low retention, clear, sterile tips 1,000 μL (Tarsons, catalog number: 527106), 200 μL (Tarsons, catalog number: 527104), and 10 μL (Tarsons, catalog number: 527100)
2. Spinwin^TM Micro Centrifuge clear, sterile tubes 1.5 mL (Tarsons, catalog number: 500010) and 2 mL (Tarsons, catalog number: 500020)
3. Dissection box
4. Spatula
5. Tissue paper
6. Kimwipes
7. Nuclease-free water (Promega, catalog number: P1193)
8. 1× sterile PBS (HiMedia, catalog number: TL1022)
9. Phenol-chloroform-isoamyl alcohol (24:24:1) (Sigma-Aldrich, catalog number: 516276)
10. Ice-cold absolute ethanol (Sigma-Aldrich, catalog number: 459836)
11. RNase A (Sigma-Aldrich, catalog number: R6513)
12. Tris-base
13. HCl
14. EDTA
15. NaCl
16. SDS
17. Milli-Q^® ultra-pure water
18. 70% ethanol (1,000 mL) (see Recipes)
19. Lysis buffer (100 mL), pH 7.2 (see Recipes)
For tissue RNA isolation
1. Filter MAXIPENSE^TM, Low retention, clear, sterile tips 1,000 μL (Tarsons, catalog number: 527106), 200 μL (Tarsons, catalog number: 527104), and 10 μL (Tarsons, catalog number: 527100)
2. Spinwin^TM Micro Centrifuge clear, sterile tubes 1.5 mL (Tarsons, catalog number: 500010) and 2 mL (Tarsons, catalog number: 500020)
3. Dissection box
4. 1 mL syringe
5. Weighing paper (Fisher Brand, size 4" × 4")
6. Tissue paper
7. Kimwipes
8. Mortar and pestle
9. RNAlater^®(Sigma-Aldrich, catalog number: R0901)
10. RNaseZAP^TM(Sigma-Aldrich, catalog number: R2020)
11. Nuclease-free water (Promega, catalog number: P1193)
12. Liquid nitrogen
13. RNeasy Mini Kit (Qiagen, catalog number: 74104)
14. DNase I (Qiagen, catalog number: 74106)
15. RDD buffer (Qiagen, catalog number: 74106)
16. 70% ethanol (1,000 mL) (see Recipes)

Equipment

Pipette (1,000 μL, 200 μL, 20 μL, 2 μL) (Thermo Fisher, Massachusetts, U.S)
-80 °C freezer (Thermo Fisher, Massachusetts, U.S)
CO₂ chamber for mice (Thermo Fisher, Massachusetts, U.S)
Vortex (Fisher Biotec, Wembley WA 6904, Australia)
Microcentrifuge (Eppendorf, Hamburg, Germany)
Laminar airflow (Thermo Fisher Scientific, Columbus, OH, USA)
Thermoshaker (Thermo Fisher, Massachusetts, U.S)
Rotospin (Tarsons, Kolkata, India) or any test tube rotator
Nanodrop 2000 machine (Thermo Fisher Scientific, Columbus, OH, USA) or any UV-VIS spectrophotometer (minimum sample volume 0–5 μL)
Qubit4 fluorometer (Invitrogen, California, USA) or any normal fluorometer
Horizontal gel electrophoresis apparatus (California, U.S)

Procedure

Part I: Microbial gDNA isolation from mouse cecal content

Collection of cecal content
1. Dissect the mouse aseptically and carefully remove the cecum.
2. Collect the cecal content in a sterile 2 mL microcentrifuge inside the laminar airflow to avoid any kind of environmental contamination. Label the sample properly. Immediately snap freeze the cecal content and store it at -80 °C for further use (Graphical abstract step 1).
  
  Graphical abstract step 1. Collection of cecal content.
Cecal DNA extraction
1. Take the cecal sample from the -80 °C freezer and keep it on ice. Aseptically transfer 180–220 mg of cecal content into a sterile 2 mL microcentrifuge tube.
2. Add 1 mL 1× sterile ice-cold PBS to the sample and homogenize it properly by vigorous vortexing and pipetting.
3. Centrifuge the suspension at 1,500 × g for 5 min at room temperature (Graphical abstract step 2).
  
  Graphical abstract step 2. Centrifugation of the cecal sample.
4. Carefully remove the supernatant. Add 600 μL of lysis buffer to the pellet and mix it properly by pipetting (Graphical abstract step 3).
  Note: Do not use vortex for mixing.
  
  Graphical abstract step 3. Lysis buffer addition.
5. Keep the mixture at 70 °C for 30 min with intermittent mixing by inverting the microcentrifuge tube several times (Graphical abstract step 4).
  
  Graphical abstract step 4. Heating at 70 °C.
6. Centrifuge the lysate at 15,700 × g for 15 min at 4 °C and collect the supernatant in a sterile 2 mL microcentrifuge tube.
7. Add 1 mL of phenol-chloroform-isoamyl alcohol (24:24:1) to the lysate and mix it properly for 2–3 min by inverting the tube approximately 12–15 times.
8. Centrifuge the mixture at 15,700 × g for 15 min at 4 °C.
9. Collect the upper aqueous phase in a new sterile 2 mL microcentrifuge tube. Repeat steps 7–9 at least twice, until the lysate is colorless.
10. Add three volumes of ice-cold absolute ethanol to the colorless lysate and gently mix by simultaneously inverting and rotating in a rotospin instrument for 5–10 min.
11. Freeze the solution at -80 °C for at least 1 h. For better precipitation of DNA, keep it in the freezer for 4–5 h (Graphical abstract step 5).
  
  Graphical abstract step 5. Precipitation at -80 °C.
12. Separate the precipitate by centrifugation at 15,700 × g for 15 min at 4 °C.
13. Wash the pellet once with 700 μL of 70% ethanol and centrifuge at 15,700 × g for 15 min at 4 °C (Graphical abstract step 6).
  
  Graphical abstract step 6. Ethanol wash.
14. Dry the pellet at room temperature for 20–30 min.
15. Add 50–100 μL nuclease-free water to the pellet and mix it with gentle tapping.
16. Add 5 μL of RNase A and keep at room temperature for 15 min.
17. Repeat steps 10–15 to remove the RNase A and recover the pure microbial gDNA (Graphical abstract step 7).
  
  Graphical abstract step 7. Microbial gDNA.
18. Immediately check the quality and quantity of the RNA, and store it in a -80 °C freezer.

Part II: RNA extraction from mouse colonic tissue

Collection of the colon tissue sample
1. Dissect the mouse aseptically and cut the colon portion from the entire gut.
2. Remove the colonic content by flushing with cold 1× sterile PBS.
3. Cut the tissue (80–120 mg) into small pieces and store it in RNALater (1,800 μL) at a -20 °C freezer for further use.
RNA extraction
For RNA isolation of the colonic tissue, we use the Qiagen RNeasy Mini Kit with an additional step of churning the tissue with liquid nitrogen for better yield.
1. Weight 20–30 mg (not more than 30 mg) of colonic tissue. Remove the remaining RNALater from the tissue sample using Kimwipes.
2. Place the tissue sample in a mortar. Add ample liquid nitrogen and immediately churn the tissue with the pestle. Ensure that all liquid nitrogen evaporates before grinding (Graphical abstract step 8).
  Note: After step 2, follow the Qiagen RNeasy Mini Kit protocol.
  
  Graphical abstract step 8. Tissue grinding.
3. Add 700 μL of RLT buffer to the mortar and mix the churned tissue with the buffer by pipetting.
4. Transfer the lysate into a 1.5 mL sterile microcentrifuge tube and centrifuge at 14,000 × g for 3 min at room temperature.
5. Carefully remove the supernatant by pipetting and add 1 volume of 70% ethanol to the lysate. Mix well by pipetting and centrifuge at 14,000 × g for 3 min at room temperature.
6. Carefully remove the supernatant and transfer up to 700 μL of the sample to an RNeasy Mini spin column placed in a 2 mL collection tube.
7. Close the lid and centrifuge at 8,000 × g for 15 s at room temperature. After centrifugation, discard the flowthrough.
8. Add 350 μL of Buffer RW1 to the RNeasy column, close lid, and centrifuge at 8,000 × g for 15 s at room temperature. After centrifugation, discard the flowthrough.
9. Add 10 μL of DNase I stock solution to 70 μL of Buffer RDD and mix by gently inverting the tube. Give a brief spin.
10. Add 80 μL of DNase I incubation mix directly to the RNeasy column membrane, and keep it at room temperature for 15 min (Graphical abstract step 9).
  
  Graphical abstract step 9. Column purification.
11. Add 350 μL of Buffer RW1 to the RNeasy spin column. Close the lid and centrifuge at 8,000 × g for 15 s at room temperature. Discard the flowthrough.
12. Add 500 μL of Buffer RPE to the RNeasy spin column. Close the lid and centrifuge at 8,000 × g for 15 s at room temperature. Discard the flowthrough.
13. Add 500 μL of Buffer RPE to the RNeasy spin column. Close the lid and centrifuge at 8,000 × g for 2 min at room temperature. Discard the flowthrough.
14. Place the RNeasy spin column in a new 2 mL collection tube. Centrifuge at full speed for 1 min to dry the membrane.
15. Place the RNeasy spin column in a new 1.5 mL collection tube.
16. Add 30–50 μL of nuclease-free water directly to the spin column membrane. Wait for 2 min.
17. Close the lid and centrifuge at 8,000 × g for 1 min at room temperature to elute the RNA.
18. Immediately check the quality and quantity, and store the RNA in a -80 °C freezer (Graphical abstract step 10).
Graphical abstract step 10. Tissue RNA.

Data analysis

The concentration and quality of the obtained DNA (approximate expected yield/concentration: 800–2,200 ng/μL) and RNA (approximate expected yield/concentration: 700–2,000 ng/μL) can be measured by NanoDrop analysis (Graphical abstract step 11). The absorbance ratios at 260 nm/230 nm and 260 nm/280 nm can be determined to evaluate the purity of the extracted DNA and RNA, respectively. DNA and RNA concentration can be validated by the Qubit fluorometer (Graphical abstract step 12). DNA and RNA integrity can be assessed by 0.8% and 2% agarose gel electrophoresis, respectively.

Graphical abstract step 11. Quantity check by Nanodrop.

Graphical abstract step 12. Quality check by Agarose gel.

Notes

It is important to collect cecal samples from all animals within an experimental cohort at the same time of the day, to avoid diurnal oscillations of the gut microbiota (Thaiss et al., 2014). It is also recommended to isolate the DNA from freshly collected samples of a single cohort at the same time to avoid storage artifacts, as freezing of cecal samples was shown to influence the ratio of Firmicutes to Bacteroidetes (Bahl et al., 2012).

Recipes

Lysis buffer for gDNA isolation (100 mL), pH 7.2
Reagent Final concentration Amount
Tris-base n/a 1.211 g
HCl 0.1M 8.940 mL
EDTA 20mM 0.744 g
NaCl 100mM 0.584 g
SDS 4% 4.000 g
Milli-Q^® ultra-pure water 100 mL Adjust the volume with water
70% ethanol (1,000 mL)
Reagent Final concentration Amount
Ethanol (absolute) 70% 700 mL
Milli-Q^® ultra-pure water n/a 300 mL

Acknowledgments

We acknowledge Dr. Biswaranjan Pradhan for helping us to standardize the DNA isolation protocol.

We also acknowledge the two previous reports from our lab by Pradhan et al. (2019 and 2022), from which we adopted the mentioned DNA isolation protocol.

Competing interests

The authors declare no competing interests.

Ethics

Committee for Control and Supervision of Experiments on Animals, Govt. of India (CPCSEA) approved this study (IAEC, Reg. No- 1634/GO/ReBi/S/12/CPCSEA, protocol number NISER/SBS/IAEC/AH-186). All experiments were performed as per the approved guidelines.

References

Bag, S., Saha, B., Mehta, O., Anbumani, D., Kumar, N., Dayal, M., Pant, A., Kumar, P., Saxena, S., Allin, K. H. et al. (2016). An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples. Sci Rep 6: 26775.
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.
Brooks, J. P., Edwards, D. J., Harwich, M. D., Jr., Rivera, M. C., Fettweis, J. M., Serrano, M. G., Reris, R. A., Sheth, N. U., Huang, B., Girerd, P., et al. (2015). The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies. BMC Microbiol 15: 66.
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.
Gryp, T., Glorieux, G., Joossens, M., Vaneechoutte, M. (2020). Comparison of five assays for DNA extraction from bacterial cells in human faecal samples. J Appl Microbiol 129(2): 378-388.
Pradhan, B., Guha, D., Naik, A.K., Banerjee, Tambat, S., Chawla, S., Senapati, S., Aich, P. (2019). Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1- and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced Diarrhea. Probiotics Antimicrob Proteins 11: 887-904.
Pradhan, S., Ray, P. and Aich, P. (2022). Microbiota transplantation from younger to older mice could restore lost immunity to effectively clear salmonella infection in Th2-biased BALB/c mice. Life Sci 288: 120201.
Thaiss, C. A., Zeevi, D., Levy, M., Zilberman-Schapira, G., Suez, J., Tengeler, A. C., Abramson, L., Katz, M. N., Korem, T., Zmora, N., et al. (2014). Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis. Cell 159(3): 514-529.
Videnska, P., Smerkova, K., Zwinsova, B., Popovici, V., Micenkova, L., Sedlar. K., Budinska, E. (2019). Stool sampling and DNA isolation kits affect DNA quality and bacterial composition following 16S rRNA gene sequencing using MiSeq Illumina platform. Sci Rep 9: 13837.