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Evaluate My Protocol: RNA Extraction w/ Fat. - (Oct/08/2009 )

Hi I'm an Undergrad and very new to RNA extraction. I follow the trizol protocol and have modified it over the weeks based on people's suggestion in the forum. So here's my current protocol.
1. Take 10 ug of frozen mice fat tissue and homogenize it with 500 mL of Trizol. I then add 500 mL vortex it (because better reagent-tissue ratio is suppose to increase yield)
2. since it's fat. I also take out 20 uL of the top layer after centrifugation in 12,200 rpm for 10 min. (too remove the extra fat layer)
3. I then put the aqueous layer into a separate tube and add 200 uL of cholroform. I then centrifuge it again at 12,200 for 15 min.
4. Then do an extra chloroform step centrifuge it for 12,200 for 10 min.
5. I take the top layer from that tube (usually about 500 uL). Transfer it to a new tube.
6. I then put in 500 uL isopropanol. Centrifuge at 12,200 for 10 min.
7. Then I usually don't get a pellet. My 1st question is, is it because the sample is too small? I have changed the aliquots of all the reagents except for trizol to counter contamination, but I still don't see a pellet.
8. Even though I don't have a pellet I still continue with the ethanol wash (2X for 5 min at 7000 rpm). I try to let it air dry for 10 min then whatever residue I try to pipet with 10 uL pipet.
9. Then viola, I spec it get a concentration ranging from 100 - 400 ug/uL and an 260/280 ranging from 1- 8. My lab technician wants it so that it's 1.8-2.1. (What is it that causes a high ratio or a low one?)
Please Help! Thanks

-MichaelM-

MichaelM on Oct 8 2009, 02:01 PM said:

1. Take 10 ug of frozen mice fat tissue and homogenize it with 500 mL of Trizol. I then add 500 mL vortex it (because better reagent-tissue ratio is suppose to increase yield)

7. Then I usually don't get a pellet. My 1st question is, is it because the sample is too small? I have changed the aliquots of all the reagents except for trizol to counter contamination, but I still don't see a pellet.
8. Even though I don't have a pellet I still continue with the ethanol wash (2X for 5 min at 7000 rpm). I try to let it air dry for 10 min then whatever residue I try to pipet with 10 uL pipet.
9. Then viola, I spec it get a concentration ranging from 100 - 400 ug/uL and an 260/280 ranging from 1- 8. My lab technician wants it so that it's 1.8-2.1. (What is it that causes a high ratio or a low one?)
Please Help! Thanks

I suspect you mean microlitres rather than mL in "1"
"7" RNA pellets can be clear and are often gel like rather than solid. Fat also doesn't have a lot of RNA in it - the cells are mostly fat with a small nucleus.
"9" Ratio is derived from the absorbance of DNA at 260 and 280 nm. Have a look at curves for DNA, protein and RNA you will see why from that, better than I can explain.

You should also refer to centrifuge speeds as RCF or g rather than RPM as this means nothing if you change the rotor size.

-bob1-