I want to extract RNA from cardiac fibroblasts. I tried both RNAzol and Qiagen kit, but the amount is quite low. I cultured it in 35mm dish, is that enough for RNA extraction. I am so confused why I cannot get the RNA. Does someone has good ideas? Thank you.
The RNA yield that you obtain depends on the amount you of cells from which you start with. If you start with low confluence, you will obtain low yields and viceversa. Assuming that you are extracting RNA from cells in high confluence you might be losing your RNA in one of the steps of purification (e.g. RNA precipitation, acquose phase recollection, etc).
Which quantification method are you using?
Always use DEPC treated plus autoclaved tips.increase the size of culture plate.
Mauricio de la Rosa-Garza
Thanks for your reply.
The FBs in my dish are quite confluent. I also thought about that some steps caused the RNA contaminated or degraded. But I tried both RNAzol and Qiagen kit
three times and found an experienced person to help me. I am sure the tips, eppendorfs, environment are clean. It's still in a low yield.
I used Nano drop to quantify the RNA.
Do you culture your FBs on the 35mm dish to get the RNA extraction or a larger dish? Is this cell type hard to get the RNA extraction? I think 35mm is enough for the Cardiomyocyte to do the RNA extraction.
Thank you
Yakun Wu
Honestly, i don't think it is a cell number (confluence) problem. I´ve tried extracting RNA from different types of samples with comercial kits (qiagen) and other "homemade" protocols. Some of sources with very low cellularity, and still get descent amounts of RNA. "Fresh" breast cancer tissue, ffpe BC tissue, whole blood and cultured cells in different formats (T25 and T75 flasks, 6 and 96 well format and 60mms petri dishes). Starting from a single 6-well plate well at an 80% confluence, i always obtain a yield of around 100ng/ul in a total volume of 15uls in the worst case scenario (using trizol).
How much RNA are you obtaining? If your are obtaining close to none, it is highly probable that there is something wrong with the reagents, the protocol or the materials used.
If someone experienced is helping/supervising you and still get low yields. The problem should reside on the methods used for extraction. Either the method used is not the best for cardiac fibroblasts or there might be something wrong with the reagents.
And yes, that plate format (35mm) should be enough to obtain RNA.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts