1,093
edits
Changes
added Trizol extraction of largish tissues
==Trizol extraction of total RNA==
J.W. Jahng, January 2000
* Use frozen tissues kept in –80 degree deepfreezer
1. Homogenize the tissue with a glass homogenizer on ice
*you can use a bucketful of ice and keep the homogenizer on the ice during homogenization.
*use 1ml of Trizol reagent for 1 adrenal gland: I think this is just about the right ratio, if you have bigger tissues, you better cut into as small pieces as 1 adrenal gland.
*homogenize the tissue until you don’t see any big chunks of tissue, don’t be scared it just takes about 10 times of gentle pestleing or so.
*you can directly pour the homogenate into the eppendorf tube, don’t use pipette.
2. Spin down at 12,000 X g for 10 min at 2-8 C.
*this step was optional on the company protocol, I think it improves your results.
3. Take the supernatant into a clean tube.
*be careful not to take the top fat layer.
*take the supernatant at once as much as possible using a P1,000 tip, and not too much worry even if you accidentally take also tiny bit of the pellet, because your chloroform will clean it.
4. Incubate the homogenized samples (the supernatant in a new tube) for 5min at 15- 30C
*permit the complete dissociation of nucleoprotein complexes.
*15-30 C means you can do it at room temperature (RT)I think.
5. Add 0.2ml of Chloroform per 1ml of Trizol
*cap the sample tubes securely.
*do under the fume hood, if you want you could do the whole procedure under the hood, but at least you use the hood whenever you add Trizol, Chloroform,etc.etc.
6. Shake the tubes vigorously by hands for 15 seconds
and incubate them at 15-30 C for 2-3 min.
*means you can just leave the tube on the bench for 2-3min at RT
7. Spin down at no more than 12,000 x g for 15 min at 2-8 C.
8. Take the colorless upper aqueous phase
*The volume of the aqueous phase is about 60% of the volume of Trizol used for homogenization.
*don’t get frustrated even if you could only take even less than 50 % (means less than 500ul eventhough you started with 1ml of Trizol), your resulting RNA will still be plenty enough. You just be careful not to take the bottom phase.
*you better use a P200 tip and collect a little by little into a clean tube to reduce the chance for contamination of the bottom phase.
9. Add 0.5ml of isopropyl alcohol per 1ml of Trizol
and incubate the samples at 15-30 C for 10 min.
*of course you want to mix the sample gently by converting the tube several times and leave it on the bench for 10 min at RT.
10. Spin down at no more than 12,000 x g for 10 min at 2-8 C.
*you’ll see white pellet in the tube.
11. Remove the supernatant and add at least 1 ml of 75 % EtOH per 1ml Trizol
*you can just pour off the supernatant, but do very gently and at once otherwise your RNA pellet might also escape from the tube. And also if you don’t decant the supernatant soon enough after spinning down(like if you leave your tube in the centrifuge even after spindown and talk with people for a while as usual), your RNA pellet may also have chance to follow the fate of the supernatant when you pour it off. In that case, don’t try to pour it off, pipette out the supernatant.
12. Mix the sample by vortexing briefly and spin down at no more than 7,500 x g for 5 min at 2-8 C.
*don’t worry even if you still see the big chunks of white pellet, it’ clean enough.
13. Air –dry or vacuum-dry for 5-10 min.
*air-dry is safer than vacuum-dry, and it takes about 20 min to get dried at RT
If you see nothing in the tube, it means it’s dried and your sample is clean.
14. Dissolve RNA in DEPC-dH2O.
*dissolve RNA with using pipette tip like pipetting the sample in-and-out a few times.
*RNA from 1 adrenal gland was dissolved with 20 ul of depc-water. and took 1ul to do OD measurement.
J.W. Jahng, January 2000
* Use frozen tissues kept in –80 degree deepfreezer
1. Homogenize the tissue with a glass homogenizer on ice
*you can use a bucketful of ice and keep the homogenizer on the ice during homogenization.
*use 1ml of Trizol reagent for 1 adrenal gland: I think this is just about the right ratio, if you have bigger tissues, you better cut into as small pieces as 1 adrenal gland.
*homogenize the tissue until you don’t see any big chunks of tissue, don’t be scared it just takes about 10 times of gentle pestleing or so.
*you can directly pour the homogenate into the eppendorf tube, don’t use pipette.
2. Spin down at 12,000 X g for 10 min at 2-8 C.
*this step was optional on the company protocol, I think it improves your results.
3. Take the supernatant into a clean tube.
*be careful not to take the top fat layer.
*take the supernatant at once as much as possible using a P1,000 tip, and not too much worry even if you accidentally take also tiny bit of the pellet, because your chloroform will clean it.
4. Incubate the homogenized samples (the supernatant in a new tube) for 5min at 15- 30C
*permit the complete dissociation of nucleoprotein complexes.
*15-30 C means you can do it at room temperature (RT)I think.
5. Add 0.2ml of Chloroform per 1ml of Trizol
*cap the sample tubes securely.
*do under the fume hood, if you want you could do the whole procedure under the hood, but at least you use the hood whenever you add Trizol, Chloroform,etc.etc.
6. Shake the tubes vigorously by hands for 15 seconds
and incubate them at 15-30 C for 2-3 min.
*means you can just leave the tube on the bench for 2-3min at RT
7. Spin down at no more than 12,000 x g for 15 min at 2-8 C.
8. Take the colorless upper aqueous phase
*The volume of the aqueous phase is about 60% of the volume of Trizol used for homogenization.
*don’t get frustrated even if you could only take even less than 50 % (means less than 500ul eventhough you started with 1ml of Trizol), your resulting RNA will still be plenty enough. You just be careful not to take the bottom phase.
*you better use a P200 tip and collect a little by little into a clean tube to reduce the chance for contamination of the bottom phase.
9. Add 0.5ml of isopropyl alcohol per 1ml of Trizol
and incubate the samples at 15-30 C for 10 min.
*of course you want to mix the sample gently by converting the tube several times and leave it on the bench for 10 min at RT.
10. Spin down at no more than 12,000 x g for 10 min at 2-8 C.
*you’ll see white pellet in the tube.
11. Remove the supernatant and add at least 1 ml of 75 % EtOH per 1ml Trizol
*you can just pour off the supernatant, but do very gently and at once otherwise your RNA pellet might also escape from the tube. And also if you don’t decant the supernatant soon enough after spinning down(like if you leave your tube in the centrifuge even after spindown and talk with people for a while as usual), your RNA pellet may also have chance to follow the fate of the supernatant when you pour it off. In that case, don’t try to pour it off, pipette out the supernatant.
12. Mix the sample by vortexing briefly and spin down at no more than 7,500 x g for 5 min at 2-8 C.
*don’t worry even if you still see the big chunks of white pellet, it’ clean enough.
13. Air –dry or vacuum-dry for 5-10 min.
*air-dry is safer than vacuum-dry, and it takes about 20 min to get dried at RT
If you see nothing in the tube, it means it’s dried and your sample is clean.
14. Dissolve RNA in DEPC-dH2O.
*dissolve RNA with using pipette tip like pipetting the sample in-and-out a few times.
*RNA from 1 adrenal gland was dissolved with 20 ul of depc-water. and took 1ul to do OD measurement.