How did you arrive to the conclusion that you have a primer dimer issue? Would be helpful to know the ladder sizes.

Yes, there is some evidence of RNA degradation given the intense band below 18s. On some gels you can see the "5s/tRNA" band, but it shouldn't be this intense. However, we do not see the smearing sometimes associated with degraded RNA.

Is the skin tissue freshly isolated? How is the RNA stored (or did you run the gel right after isolation?).

It's also vital that all equipment should be RNase free and that the water is RNase-free.

Hi, I don't have experience with skin but I know is a tough tissue. I find some interesting discussion on research gate, in the first they post a protocol.

https://www.researchgate.net/post/Can_anyone_suggest_a_protocol_for_RNA_extraction_from_mouse_skin

https://www.researchgate.net/post/What_is_the_best_method_to_disrupt_and_homogenize_skin_punch_biopsy_samples

https://www.researchgate.net/post/Troubles_with_RNA_extraction_from_mouse_s

Hello, Can Kiessling

Thank you for your answer.

I used 1Kb ladder and my GAPDH is 160 bp.

i used both freshly isolated skin and frozen skin , and I run the RNA right after isolation.I get the same results for both samples. I also use DEPC water.

What is the increments for the 1 kb ladder because i see 11 increments thus leading me to believe it is not in multiples of 10 (it is not 100 bps, 200 bps, etc?). Is the lowest band 100 bp? Are you able to run a single sample on a bioanalyzer? The results for that would present a more compelling evidence of degradation.

Hi, from from my experience with fibrous (muscle) tissue, using liquid nitrogen method it is very demanding. You need strenght and you have to pay attention to not take damage to your hands, when you have to be rapid to not melt your tissue powder. Did you pour the tissue powder directly in the tub ewiht trizol or did you use a spatula to move your sample in the tube? Did you cooled the spatula if it the case?

I add an extra centrifuge after I lysed my tissue in trizol, before adding clorophorm to eliminate some fat and fibrous part. For example you can try this other centrifugation after the needles step. If the RNA recovery is so low you can add sterile glycogen to the isopropanol step.

To improve the quality of the gel image wash with NaOH your gel apparatus and the equipment in particular if it used also for PCR by you and by your coworkers. Never use the apparatus for checking RNA, after is is used for DNA analisis, without first washing it really well.

PS. Please use extra gloves, don’t process more that 3-4 samples at time, ask for help if needed, working with liquid nitrogen is dangerous

if it is not a problem with my PC, you may have a little protein contamination (the wells were not clean), check after washing the apparatus, maybe it is only a cleaning problem

From the links that I posted above it is a common problem to have low RNA recovery from skin extraction

Good luck and take care

Fatuma.

You should check every steps even small things.

First check your primer sequence (GAPDH).

- Is this a universal primer or you designed it?

- Direction of primer (5'-3') is correct?

- Can you order another GAPDH primers amplifying other region.

* I have another set of GAPDH primers, if you need, let me know.

Second, cDNV conversion.

- After conversion, PCR with another universal RT primers, such as XBP-1, CHOP, IL-1b etc. If you get the clear one band, your protocol is fine, but not you should check the cDNA construction protocol and kit.

Good luck to you.

- Same. I have several primer set, let me know, I can share it with you.

HoSeong

Dr HoSeong,

Thank you for your answer.

The first problem is RNA isolation, i found that with all the trials my RNA extraction is failed with high amount of RNA degradation.I need to overcome RNA degradation for further experiment.

Hello, Can Kiessling.

Yes the lowest band is 100bp.

I don't run a single sample on a bioanalyzer, no bioanalyzer in our lab.

Thank you for your concern.