- How do primary cultures of mouse luteal cells obtain enough total RNA for qPCR experiments? The basic procedure of my experiment is to isolate the luteal cells and seed them in a 24-well plate according to 1-2*10^5 cells. Four hours later, the cells are attached to the wall and changed the medium. After being cultured for about 12 hours, the cells are treated with drugs for 48 hours, and the cells are collected for qPCR experiments? Thanks for the help
Dear Jia,
How much hours did you incubate your cells with the lysis buffer?
Hello Jia Zhang
If you have good techniques available, you can reliably perform RT-qPCR even on a sample size of about 100 cells. If the number of cells is small, you may find it difficult to see the pellet while isolating RNA using TRIzol. I would suggest you use glycogen to co-precipitate the pellet into something more visible. You may add glycogen prior to the isopropanol to precipitate your nucleic acid.
Also, do not add extra TRIzol as adding extra TRIzol will dilute your concentration of nucleic acid, which will make precipitation harder. Using less amount of TRIzol is also not recommended as it could result in incomplete lysis.
You may add 0.3–0.4 mL of TRIzol reagent per 1 × 10^5—10^7 cells directly to the well of the 24-well plate to lyse the cells. Pipet the lysate up and down several times to homogenize.
Hope you obtain enough RNA for your qPCR experiments.
Good Luck!
some cell types are harder to lyse and using a lysis buffer in the well then adding trizol could improve yield.
Mohamed Khashan Thank you. I usually lyse cells for one minute.
Malcolm Nobre Thank you. First, we tried to use Trizol to lysis cells, but found that the quality of RNA obtained was not very good, so we used Qiagen RNeasy micro kit (74004) to extract RNA, but still very little RNA was extracted.
Julie Ann Dougherty Thanks. Maybe I can try.
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