Dear Researcher
I am working on passion fruit peel at ripening stages, and want to do qPCR but facing problem in getting good quality RNA from fruit peel,
I have tried to extract the RNA from the ripened fruit by using different RNA extraction kit from different companies, such as Qiagen, Takara, Bioflux, Genestar, and but the concentration is about 100-200 ul/ml, with very bad quality is such as A260/A230 = 1.3-1.60,
but by Trizol method, the concentration was about 600-800 ul/ml, but the quality was the same such as 1.3-1.70,
As I picked the sample from field myself 1 -1.5 months ago and transferred to Lab by keeping in 10 C, thermobox, and then peeled, dried into liquid nitrogen and kept -80 C,
while extraction, i have tried by taking the (powder) sample from 50 -200mg but all have same results,
looking for your suggestions,
I have a experience to use TIANGEN company kit for extracting RNA from Strawberry fruits. So far as I know this kit is very good and i got very good quality RNA for RNA sequencing. Actually RNA quality depends on different factor including sample collection, preservation and extraction process. Immediately after sample collection, sample must be place in Liquid nitrogen, preserved in -80 degree centigrade, for sample powdering mortar pestle should be heated at least 3-4 hours at 180 degree centigrade, use all sterilize RNA free tube and pipette tip, working area must be sterilized, working area must be isolated from contamination. You can try TIANGEN Kit.
Santosh Kumar Bose thanks sir for reply
i have tried Tiangen also but same results
Hi Hafiz Muhammad Rizwan ,
Try RNA precipitation with ammonium acetate in your already extracted samples to clean at 230nm:
Protocol: http://www.genelink.com/Literature/ps/PS40-5132_PptionSoln_Ver3.1.pdf .
There is a detail in this protocol:
1. Add 0.5 volume of 7.5 M Ammonium Acetate. Vortex.
2. Centrifuge at 12 K rpm for a minimum of 15 minutes at 4°C . TAKE THE SUPERNATANT
3. Add 2 volumes IN THE SUPERNATANT! of 100% ethanol for DNA or 2.5 volumes of 100% ethanol for RNA .
Best,
Jacó
- Badges
- Science method