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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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RNA isolation problems
Posted by: sgly005 (IP Hidden, Unregistered user, )
Date: January 24, 2005 01:12PM
I am trying to isolate RNA from approx. 100mgs of heart tissue using the Qiagen RNeasy MIDI kit. I am also using proteinase k as reccommended by Qiagen for fibrous tissue. Unfortunately my yields are very low and I am not sure why. Is there anything I can do to increase my yield?
Re: RNA isolation problems
Posted by: mamuller (IP Hidden, Junior member, 10)
Date: January 31, 2005 03:10AM
Use GeBAflex-tube RNA GEL extraction protocol, at bottom of page of www.geba.org
Re: RNA isolation problems
Posted by: Boby. T.Edwin (IP Hidden, Unregistered user, )
Date: February 14, 2005 11:26AM
SIR, Iam focussing on the pathological issues of coconut. For a differential display I am in need of RNA from mature leaf samples. I am following GTC method. The RNA on gel analysis appeared as degraded. I would like to know 1. whether the RNA in mature leaf tissues are in degraded form. ? 2. Should I have to do DNAse treatment befor attempting for RT? 3. What are the possible reasons for degraded RNA? 4. Please suggest a simple and effective method for isolating RNA from mature plant tisses? Kindly mail to me the answers to these questions at the earliest so that I can continue my research. Yours faithfully Boby
Re: RNA isolation problems
Posted by: ranadheer kumar (IP Hidden, Unregistered user, )
Date: February 14, 2005 09:50PM
hi i am ranadheer,
i don't think the RNA in matured leaves will be in degraded form. as soon as you grind the tissue don't allow it to thaw,immediately add GTC solution(containing beta mercapto ethanol)./ i hope it will reduce the chance of degradation. if you want i can search in Qiagen protocols, there you can find the complete info about handling and all. bye ranadheer
Re: RNA isolation problems
Posted by: priyanka singh (IP Hidden, Unregistered user, )
Date: May 11, 2005 10:24AM
hi
i am extracting RNA from head tissues using trizol and i get good pellet .but this pellet is pinkish instead of white.i am taking full care of not taking the interphase while taking the upper aqueous phase but still the pellet carries color. please suggest how can i get white pellet. thanx
Re: RNA isolation problems
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 11, 2005 01:22PM
i have gotten pinkish pellets before and it usually results from phenol contamination. An easy to remove the phenol is to do an additional wash in 75% ethanol. To check for phenol contamination assay the absorbance of your samples. If contamination with the aqueous phase has occurred the A260/280 ratio will be less than 1.6.
RNA isolation problems
Posted by: priyanka singh (IP Hidden, Unregistered user, )
Date: May 16, 2005 11:28PM
thanx a lot for reply..i also want to know if precipitating RNA at -20 degrees (in isopropanol) gives you better yields than at room temp.
Re: RNA isolation problems
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 17, 2005 11:15AM
from my experience ice cold isopropanol can give better yields than at RT.
Re: RNA isolation problems
Posted by: siRNA (IP Hidden, New member, 1)
Date: May 24, 2005 10:13PM
Sometimes it takes me 2-3 hours at -20 C to get decent precipitation. From my experience precipitation at -80 C for 45 minutes does the trick
Re: RNA isolation problems
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 25, 2005 12:17PM
siRNA, i never had to wait that long to precipitate RNA using the Trizol method. the recommended incubation time at room temperature is only ten minutes.
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