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RNA extraction from human jaw bone

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#1 Aom

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Posted 17 August 2013 - 08:42 AM

I've tried to extract RNA from the human jaw bone, very hard and minimal amount of cells, The yields were very low (the best RIN got from the bioanalyzer was about 4/10 ). I submerged the samples in RNAlater then used the cryogenic grinder for griding the bone particle, it took about 10 minutes per 3 samples for transfering the samples between the miller tubes. The RNA isolation kit was Qiagen RNeasy lipid tissue kit with the spin columns. Could anyone tell me how to improve the RIN to be good enough for Microarray or qPCR ?

Would it be better if I used TRIZOL with Purelink rna kit ?

 

Thank you in advance,

Aom



#2 Curtis

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Posted 18 August 2013 - 07:55 PM

Trizol always gives higher amount of RNA, but you might have lower purity. How much is your concentration? I'm not sure how much '4/10 by bioanalayzer' is exactly. Usually column-based extraction has low yield, but since you are dealing with a particle you must have a lot of RNA.



#3 Aom

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Posted 19 August 2013 - 12:14 AM

I've got concentration < 70 ng/ul  with A260/A280 ratio ~ 1.5-1.8 . 

I've used Qiazol from the Qiagen kit, but not sure if Trizol would be more efficient .



#4 Curtis

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Posted 19 August 2013 - 12:20 AM

I don't think they are very different. Only the method is different. Trizol method pellets down RNA in the last step. But this concentration is not as bad as you say.



#5 PhalanxBio

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Posted 19 August 2013 - 08:30 AM

Hello.  I think one problem with your protocol may be related to your use of RNAlater.  RNAlater does indeed preserve RNA integrity but it usually takes some time for the RNAlater to penetrate tissue.  Proper use of RNAlater is to use thin tissue specimens and let them sit in RNAlater overnight at 4 degrees C then transfer them to -20 or -80 for longer term storage.  RNA isolation from RNAlater-preserved samples is not straightforward as the high salt concentration can be incompatible with other isolation buffers.  Removing the RNAlater before isolating the RNA is very important.  I suggest you use RNA lysis buffer in place of RNA later.  Using this method, your tissue will be homogenized inside RNA lysis buffer instead of RNA later.  In my experience, tissue homogenization usually occurs inside of lysis buffer.  Hope this helps!







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