Hello all,
I have recently gone through the Illumina Trueseq RNA sample prep kit with no success. Has anyone else had problems with this kit/what were any problems encountered?
A little bit more about what I did:
I extracted with Ambion's RNAqueous 4-PCR kit but do not have a bioanalyzer to assess total RNA quality prior to the protocol which I have heard can have a huge effect on yields. Instead I ran a 2% agarose gel in TAE and stained with Ethidium. I got one bright, non-smeared band (I am working on Lepidopteran species which have a "hidden-break" in the 28S band, so it migrates with the 18S band) for all of my samples, so I assumed the RNA was ok. I ran 10 uL of sample on this gel with RNA concentration being from .332-1.66 ug/mL.
Questions:
1) Would it have been better to run a denaturing RNA gel? I talked to Illumina tech support and they recommended this. Could I see denaturation better?
2) I standardized the total RNA put into the Trueseq protocol to 1 ug... should I have used more starting material? This is on the lower end of the recommended range of .01-4ug.
3) Our department just acquired a nanodrop so I speced the RNA that had been stored at -20C for 2 weeks since I tried the Truseq protocol. The 260/280 readings were from 1.25-2.03. I don't have a lot of experience with these machines but think the range I am looking for is 1.8-2.1 , does this mean that some of my samples are contaminated with excess salts/proteins/something else?
Any advice would be appreciated. I know crap in is crap out, so does it seem like my starting material is ok/are there any additional methods I can use to check it?
Thank you!
I have recently gone through the Illumina Trueseq RNA sample prep kit with no success. Has anyone else had problems with this kit/what were any problems encountered?
A little bit more about what I did:
I extracted with Ambion's RNAqueous 4-PCR kit but do not have a bioanalyzer to assess total RNA quality prior to the protocol which I have heard can have a huge effect on yields. Instead I ran a 2% agarose gel in TAE and stained with Ethidium. I got one bright, non-smeared band (I am working on Lepidopteran species which have a "hidden-break" in the 28S band, so it migrates with the 18S band) for all of my samples, so I assumed the RNA was ok. I ran 10 uL of sample on this gel with RNA concentration being from .332-1.66 ug/mL.
Questions:
1) Would it have been better to run a denaturing RNA gel? I talked to Illumina tech support and they recommended this. Could I see denaturation better?
2) I standardized the total RNA put into the Trueseq protocol to 1 ug... should I have used more starting material? This is on the lower end of the recommended range of .01-4ug.
3) Our department just acquired a nanodrop so I speced the RNA that had been stored at -20C for 2 weeks since I tried the Truseq protocol. The 260/280 readings were from 1.25-2.03. I don't have a lot of experience with these machines but think the range I am looking for is 1.8-2.1 , does this mean that some of my samples are contaminated with excess salts/proteins/something else?
Any advice would be appreciated. I know crap in is crap out, so does it seem like my starting material is ok/are there any additional methods I can use to check it?
Thank you!
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