We are trying for both RNA and DNA extraction for sequencing from the microglia cells (single cells) isolated from a single adult mouse brain. Currently, we are using MACS miltenyi CD11b microglia microbeads to isolate purified microglia, followed by QIAGEN AllPrep simultaneous DNA -RNA extraction kit for DNA -RNA extraction but unfortunately I am not getting enough quantity and good quality RNA for RNA seq analysis. Sometimes I am getting DNA (quantity and quality enough) using QIAGEN AllPrep kit that is enough for DNA seq assay. Can you please suggest me any protocol or kit or any modification so that I can increase my extracted RNA yield (both quality and quantity, #500ng/12ul and RIN>8 is my requirements for RNA seq), Thanks for your time and expertise in advance.
Is the brain tissue frozen tissue? If so, during freeze/thaw the plasma membrane can rupture and cause RNA loss which may be leading to your low yield. You can try single nuclei isolation/sequencing to avoid this since the nuclear membrane stays intact during freezing. I recommend the single nuclei isolation kit for neuronal tissue from Invent Biotechnolgies for this. It has a high yield and avoids nuclei clumping, and also separates myelin to prevent contamination.
https://inventbiotech.com/products/minute%E2%84%A2-single-nucleus-isolation-kit-for-neuronal-tissues-cells-20-preps?_pos=1&_sid=9f8e5b0ae&_ss=r
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