I had to perform RNA extraction from the mice skin sample (shaved and hair removed tissue sample of about 1*1 cm square). For that I pulverized the tissue in liquid nitrogen pre-chilled mortar and pistle and extracted RNA with Trisol reagent (Trisol-chloroform-isopropanol method), general method for RNA extraction. While quantification, I got sufficient RNA with good quality (I mean 260:280 ration is >1.9 -2 and 260:230 is >1.5 in each sample. I made cDNA (using 1 microgram of this RNA) and performed the qRT-PCR. Surprisingly some sample Ct value is not detected and other also have the quite high variation in Ct value. I tried with different house keeping genes (HKG) (actin, GAPDH, CycloQ, 18s etc In HKG also there is variations in Ct value in among normal mouse (eg. Ct value ranges from 14-19 form normal group). However, according to in many researches there was no problem with the RNA extraction methods in my case, I am unanswered that where is the wrong gone? For other tissues, such as brain, I got perfect results (almost same Ct value for normal group with HKG). Could you please suggest me where is the problem in my case? I will be happy to get the answer from the researcher who did the RNA -qRT-PCR in skin sample (experienced one).
Did you check your RNA profile on a gel/bioanalyzer. Was RNA from brain etc that gave positive results isolated at the same time or was the RT performed at the same time? Maybe some impurities in RNA that inhibit your RT reaction...
You mentioned pulverizing the tissue. Was this efficient enough, what was the yield? To isolate RNA from skin, we normally use cryosectioning (50 pieces at 20 µm). To dissociate we add beads and Trizol (1 ml of 200 mg tissue) and use a bead-beater (5 min 40 Hz).
Are you interested in the dermal or epidermal cells?
Thank you for Dr. Bouke and Kartiki for the suggestion. Dr. Bouke, I think pulverizing is sufficient, because the yield was high enough that is, ~800 ng/microliter. At the moment I am not separating the dermis and epidermis. But haven't use bead-beater yet. Is there any other method beside this?
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