How can I best collect samples from the small intestinal (ileal) microbiota of neonatal mice (e.g. postnatal day 3 or 7) for 16S amplicon sequencing? The neonatal intestine is very thin and ruptures easily, so it is almost impossible to squeeze out its contents as done for adult mice. People have published to just snap freeze the whole intestine including the host tissue and extract DNA from there but I am worried that the 16S amplicon PCR will be inhibited by the huge excess of eukaryotic DNA. Other people have published to scrape out the contents of the opened intestine but again I am a bit worried about the fragile tissue. In a third paper, the authors had to pool 6-8 mice per time point because there wasn't enough material to sequence individual contents. So, could anybody recommend a protocol for the isolation of ileal contents of neonatal mice? Thanks!
I would open intestine to avoid tissue contamination with host DNA (as much as it is possible) but if you use more mice they might differ in their microbiota
Not sure this will be helpful at all, but I'll throw this out there.
How are you doing it now?
We don't use neonates in our lab, but we do use lots of intestines... What we do is remove the intestine from stomach to rectum, place it in a dish with a liquid to keep it moist/ pliable, and then carefully cut the section of interest. Then, hold the section with forceps in the left hand and use a second set of forceps to carefully and slowly squeeze out the contents. If squeezing is too problematic, you can also use a needle and syringe to flush out the entire contents into a tube. (at that age, the stool should be pretty soft). Just use a small gauge and insert the needle vertically into the lumen, between the ends of the forceps, while you are holding it up. I think that keeping the tissue moist is key more than anything in preventing it from tearing.
Thank you both for your helpful answers.
I indeed separated the contents as much from the host tissue as possible and the PCR worked just fine even for day 3 using individual mice (no pooling required). I used a forceps to carefully squeeze out the colon in one piece and the ileum in 3-4 pieces of 1 cm length each.
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