I have done head kidney (HK) primary cell culture from Atlantic salmon (post smolt) before. I use 1.5 ml TRI reagent (Sigma) to extract RNA from 2 million primary HK leukocytes, following DNase treatment (Ambion) and reverse transcription for cDNA, this method gave me good Cp value from housekeeping gene (EF-1a and b-actin, Cp=12~13). Hundreds of samples were done and no problems were observed.
However, I did the same procedure in primary spleen cells (I used percoll to separate from red blood cell, wash 3 times with fresh medium after centrifuge) and I got EF-1a Cp= 20. I also obeserved 1 million cells gave me the best cDNA (EF-1a=20), but it went worse when I put more or less cells (4,2, 0.5, 0.25 million). But I can obtain good Cp value (EF-1a = 12) from the spleen tissue sample (also salt water salmon) in RNAlater (also use TRI reagent).
Now I will first try RNA extraction kit from Bioline and see how it goes with kit compare to TRI reagent method. I am wondering if anyone have similar experience and could suggest me what can I do to improve my cDNA quality.
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