I'm trying to extract RNA from superiori cervical ganglia in P2 mice. I obtained very few RNA, so I'm wondering if it's because the samples are very very small, or if it's due to technical issues. I will be doing it again in the next week, but in wainting for new pups, has anyone already tried this, and what amount of RNA did you obtain ?
Thanks a lot for your help !
An SCG from P2 mice is about 2mm length and 1mm wide. I would like to do RT-PCR followed by qPCR
Hi Emilie, I think you have a combination of the two things. The tissue is indeed very small and also not very dense of cells. I haven't specifically worked with it, but from a pituitary or a striatum or a hypothalamus of so young mice I extract between 10 and 50 ug of RNA. But you might also have a technical problem if you are using a column based extraction method. Columns always give a yield below 60-50%. I would not recommend a trizol based extraction either, since you will never be able to sufficiently dilute the guanidine isothiocyanate with such small amounts of RNA and this will unpredictably affect your RT reaction later on. Best is to do a simple cytoplasmic lysis of your tissue, douncing it in a very simple buffer (100 mM NaCl, 50 mM Tris/HCl pH 7.4, 10 mM MgCl2, 1% NP-40 or Triton and 1-2 u of RNAseIn or similar), so to minimize gDNA contamination. Then spin to remove nuclei and transfer the supernatant in a new tube and bring to 1% SDS and 0.5 mg/ml proteinase K. Digest 3-4 hrs at 37 °C and then simply extract with phenol/CHCl3 and Ethanol precipitate. Treat with DNAse and re-extract, this time also twice with CHCl3 alone, to remove all traces of phenol that would disturb the RT. After Ethanol precipitation resuspend in a small volume, 20 ul for example and check your RNA at the Nanodrop for its chemical quality and on a 1.5% agarose gel (0.5-1 ug will suffice, also in TAE, running short, just 1-2 cm) for its integrity and real quantity (you should see the two ribosomal RNA bands clearly, in a ratio of at least 2:1, and keep in mind it will be less than the Nanodrop might make you think because you always have small pieces of degraded RNA and DNA that on gel you will not see... normally the real amount of intact RNA is 30% less than you quantify by the Nanodrop). If you have a Bioanalyser, even better!!
Good luck!