There's not enough information here to diagnose the problem so I'm going to answer your question with more questions, sorry!

How much tissue was used and how was it homogenised?

What is the elution volume of these samples (how much of the 20ng/uL RNA solution do you have?)

What are the actual ratios you're getting?

What do you want to do with the extracted RNA?

8-10 mg tissue. I homogenized by using Qiazol. elution volume is 60 Ul RNA volume. 260/280 is 1.7 but 269/230 is very low 0.87. I want to perform RT-qpcr.

Ok so if we calculate the actual yields

5ng/uL * 60uL = 300ng

20ng/uL * 60uL = 1200ng

These are on the low side for 8-10mg of tissue but not shockingly low. My best guess is that the homogenisation/lysis is the problem. When you say you homogenised by using Qiazol, what did you actually do to disrupt the tissue so the RNA would come out?

I agree with you. Well, I usually add 700 ul of Qiazol into the Eppendorf tube containing the ovary sample and start homogenization. It takes sometimes 30 to 50 seconds to completely disrupt the tissue. Is there time is a factor? Later someone suggested putting the sample on ice during homogenization. I did accordingly. But no change.

I still don't understand what you are doing to homogenise the tissue. Qiazol does not magically melt the tissue, are you using a bead beater? sonicating? crushing/grinding? vortexing?

Laura Leighton No, I am just homogenate with a homogenizer no bead beater, no crushing/grinding/ and even no vortexing. our previous lab members used the same method to extract RNA. But I am failing over and over.

What kind of homogeniser? Is it glass dounce tubes like this? https://en.wikipedia.org/wiki/Dounce_homogenizer

An electric instrument with a probe you stick directly into the sample (rotor-stator homogeniser)? An electric instrument that you put the tubes in and it shakes them? A pellet pestle?

I'm sorry I know these are probably annoying follow up questions but the kind of failure you're experiencing sounds like it could be caused by inefficient disruption of tissue, resulting in the RNA basically not coming out and being thrown out with the tissue fragments. But I honestly cannot understand from your answers what you are doing to disrupt the tissue. There are maybe a dozen different items or instruments that can be called 'a homogeniser', some work way better than others, and all of them involve some kind of crushing, grinding, blending or shaking. Could you please be as specific as possible about what happens to turn your chunk of tissue into not-a-chunk-of-tissue-anymore?

No, it's completely okay to figure out any problem. I am fine if you ask a question. Here is the link to the homogenizer I use to disrupt the tissue. You can have a look

https://www.labindiainstruments.com/omni-international-laboratory-homogenizers.html .

Well, My sample was kept at -80°C. I brought it out and kept it on dry ice. Then I add 700 ul of Qiazol and strat homogenization. my protocol says max 10-20 sec homogenization but even after 20 sec the tissue wasn't disrupted completely. So I did homogenization again for 20 sec to completely lysis the tissue. My question is, if I do longer homogenization, is there any chance to degrade the RNA and yield become very low?

Mohmaad Mahmud Hossain

Just sharing some tips from my experience:

1. Ensure the tip of the homogenizer is gently touching the tissue sample.

2. Allow a gap of 5-10 seconds between each 20-second grinding session. This helps reduce the heat generated during the process. I tried up to 60 seconds with time gaps and achieved good concentration.

3. I found that keeping the tissue on dry ice made it too hard to grind. Instead of dry ice, consider using a convenient -20°C or 0°C mobile freezer.

Anmi Jose Thanks for your advice. It works for me. Thanks Once again. Highly appreciated. !!