I am using column based RNA extraction Kit. I took 20 mg testicular tissue and perform snap freeze in liquid nitrogen, after that homogenize in trizol with syringe and follow the kit protocol. Final RNA concentration is between 300 to 400 ng/ul. The 280/260 ratio is 1.8. Is that good concentration or my procedure have errors?
To determine whether your yield is good, we need to know the total amount of RNA you isolated (ie, 300-400ng/uL in what volume?)
From mouse brain tissue I routinely get 20 micrograms of RNA from 20mg of tissue. This is using a column kit, which yields somewhat lower than trizol. I did extract from testis a few times using trizol and I remember being amazed by how high the yield was, it seems like this tissue is very rich in RNA.
Dear Masud Imani,
The concentration of extracted RNA is not a very important criterion, but the quantity and quality parameters are important. The amount of RNA extracted, which showed a light absorption ratio of 260/280 equal 1.8, is excellent because concentrations above 2 and below 1.8 are signs of DNA and protein contamination, respectively. Low concentrations of RNA can also produce high concentrations of cDNA. The important point is that you should evaluate the quality of the extracted RNA with 1% agarose gel to see the 28s and 18s ribosome bands for confirmation.
Best Wishes.
@ Laura Leighton Thanks a lot for your attention,Final volume is 50 miclit, approximately totally 15 to 20 micrograms yield.
yes i agree with you that testicular tissue a cellular tissue and i expected that more than 2000ng/ul was yield. I guess that my homogenization protocol with syringe do not release RNA's completely from cells. Do this problem impair downstream to qPCR? Can i use current RNA extracted for cDNA synthesis?
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue