Hello,
I have formalin-fixed paraffin-embedded samples of breast cancer and I want to extract RNA from them. I tried several times with the RNeasy FFPE Qiagen extraction kit, but I usually get low RNA concentration (~10-20), and also the 260/280 ratio never reaches 2.0. Moreove, the 260/230 ratio is usually ~0.8.
I really don know what am I doing worng? So please if anyone could help I will be grateful.
I tried to cut my samples by a microtome and tried to use a scalpel and there is no difference.
thank you.
Hi Manar Serageldin,
I've been facing the same problem too but with a different kit called Macherey-Nagel Total RNA Isolation from FFPE. I was trying to extract RNA from formalin-fixed paraffin-embedded samples of colorectal cancer. For the first few attempts in extracting RNA from the FFPE tissue, I followed exactly what the protocols say, where it stated to incubate the FFPE with proteinase K in the lyse step for only 15 mins which gave me very low RNA concentration (~10-40), and also the 260/280 ratio. However, after troubleshooting I got to know that we can optimize the duration of incubation from 15 mins up to 3 hours with proteinase K (overnight digestion can also be done, but up to 3 hours is the optimal digestion time) depending on the size and thickness of samples and also by looking at the portion of the undigested sample. So I started to incubate my samples for approximately 2 hours and I managed to get a high amount of RNA concentration (~500-1500). However, for the 260/280 ratio, the best I have gotten to date is 1.9. Maybe you should try to increase the washing steps to get a good ratio of 260/280 and 260/230.
I hope this helps.
All the best to you.
I've also gotten poor performance from the miRNeasy FFPE kit on microdissected tumors. I had previously used the Qiagen AllPrep DNA/RNA FFPE Kit during a pilot experiment and got much better yields. My 260/280 ratios were around 1.7, but my 260/230 ratios were only around 0.4. These samples were used for RNAseq with better than anticipated performance given that the starting material was a tiny piece of FFPE tissue.
Hi Sarminiyy Lenga Gururuloo
Thank you for your reply. The protocol states that I should add proteinase k and incubate the sample for 15 mins at 56’C and 15 mins at 80’C. Do you think I should increase the incubation period at room temperature first? Or the 56’C or 80’C incubation period?
And for the samples, how did you cut your samples? Using a microtome or by a scalpel?
Hi Manar Serageldin ,
According to my protocol, I only incubate my sample with proteinase K at 56'C. Plus, proteinase K enzyme activity increases at 56'C. Most probably, the second incubation at 80'C in your protocol is to inactivate the enzyme activity. So, I would suggest you to increase the incubation period at 56'C. Hope this works for you.
And for my samples, I used microtome. Thanks and all the best.
Hi Sarminiyy Lenga Gururuloo ,
I really want to thank you. Increasing the incubation period at 56'C worked with my samples. Wishing you the best.
That's great! Hope everything goes well for you. All the best!
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