I extracted RNA from my cells cultured in soft agar culture system by TRIZOL, but unfortunately pollution in RNA yield is high so I couldn't detect genes expression by real time PCR. Any one has a solution for this problem?
thank you
Hi,
Before answering your question, I think it is important for you to provide more details about your RNA sample:
Generally, Trizol extraction is quite efficient at removing proteins and lipids as long as you collect the aqueous phase without agitating other phases.
Hi Dear Hong Sheng Cheng
My RNA yield is almost 100-120 ng/ml but its ratio in 260/280 and 260/230 is not appropriate and for example they are 1.55 and 0.36 respectively. so I think, this low OD is a obstacle for detecting genes expression by real-time PCR. Also I collect aqueous phase without agitating other phases.
Hi,
Based on the information you provided, I agree that your sample is highly contaminated. One of the main culprits of your problem could be phenol, which is a PCR inhibitor. It absorbs at about 230nm and 270nm, which may explain your low OD ratios. Furthermore, it is also very likely that the RNA yield of your sample was overestimated because contaminants could also absorb at 260nm.
Personally, after Trizol extraction, I will follow up with column-based kit (Qiagen RNeasy kit) to clean up the RNA samples. This almost always works well in my hands. In your case, you may try to reprecipitate your sample. Please check this thread for more information (https://www.researchgate.net/post/What_is_the_effect_of_the_260_280_value_on_PCR).
I hope this helps.
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