I need to extract total RNA from rat different tissues with trizol to do the qRT-PCR. I have tried twice. but, I could not get the high-quality RNA, this is my protocol, anyone can help me check if there are some problems with my protocol?
1. Place a 0.5 mm(millimeter) glass bead in tube.
2. In a chemical fume hood, add 1 mL of TRIzol reagent to a tube and place it on wet ice.
3. Put frozen tissue into tubes containing TRIzol.
4. Homogenize the samples for 1 minute at 6m/s. If the sample is not fully homogenized, repeat for no more than 2 additional minutes.
6. Working in a chemical fume hood, transfer the homogenized sample by pipet to a new 2 mL tube. Invert tube to mix. Place the tube on wet ice.
B. Phase Separation
1. Incubate the homogenized sample for 5 minutes at room temperature.
2. In a chemical fume hood, add 0.2 mL of chloroform per 1 mL of TRIzol reagent.
3. Cap sample tubes securely and shake vigorously by hand for 15 seconds. Incubate the samples at room temperature for 3 minutes.
4. Following the incubation, centrifuge the samples at 11,600 x g for 15 minutes at 4°C.
5. Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, an interphase and a colorless, upper aqueous phase. remove the upper aqueous phase to new tube.
C. RNA Precipitation
1. Add 0.5 mL of isopropanol, pipetting up and down gently. (Do not vortex.)
2. Incubate the samples for 10 minutes at room temperature, then centrifuge at 11,600 x g for 10 minutes at 4°C.
3. The RNA precipitate forms a translucent gel-like pellet on the side and bottom of the tube.
D. RNA Wash
1. Add 1 mL of 75% cold ethanol.
3. Mix by inversion. Do not vortex. Centrifuge at 10,000 x g for 5 minutes at 4°C.
E. Resuspending the RNA
1. Remove supernatant with a pipet. Air dry the RNA pellet by leaving the tubes open on the counter for approximately 15-30 minutes.
2. Resuspend the RNA pellet in 50 µL of nuclease-free water by pipetting up and down gently and incubating in a 60°C water bath for 10 minutes.
F. DNase Treatment of RNA
1. Add 5 µL) of 10X DNase I buffer and 1 µL of DNase I (2 units) to the RNA. Mix gently and incubate in a 37℃ water bath for 20 minutes.
2. 75℃ 15min.
Test the concentration
Check RNA Integrity with 1% aerogel
Hi, Lili Hou
I’ve been doing RNA extractions for a long while, from a lot of different tissues in different species, like fish, oysters, mussels, frogs, shrimps, whales, dolphins, birds, and recently, nasopharyngeal samples for covid diagnosis. The latest was by bead extractions. All the others with phenol/chloroform protocols.
In these samples, we use the same protocol with few alterations in the amount of tissue processed.
You can use beads, pestle, or manual homogenizers like tissue tearor.
Our protocol is the same that you are using, with few modifications as follows (I use your protocol to point the alterations in bold type).
1. Place a 0.5 mm(millimeter) glass bead in tube.
2. In a chemical fume hood, add 1 mL of TRIzol reagent to a tube and place it on wet ice.
3. Put frozen tissue into tubes containing TRIzol (50mg of tissue per 1 mL of Trizol).
4. Homogenize the samples for 1 minute at 6m/s. If the sample is not fully homogenized, repeat for no more than 2 additional minutes. (We process the sample until fully homogenized)
6. Working in a chemical fume hood, transfer the homogenized sample by pipet to a new 2 mL tube. Invert tube to mix. Place the tube on wet ice.
B. Phase Separation
1. Incubate the homogenized sample for 30 minutes at room temperature or in a heat block at 20 ºC.
2. In a chemical fume hood, add 0.2 mL of chloroform per 1 mL of TRIzol reagent.
3. Cap sample tubes securely and shake vigorously by vortex for 15 seconds. Incubate the samples at room temperature for 3 minutes.
4. Following the incubation, centrifuge the samples at 14,000 x g for 30 minutes at 4°C.
5. Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase, and a colorless, upper aqueous phase. remove the upper aqueous phase to a new 1,5 mL tube.
C. RNA Precipitation
1. Add 0.5 mL of isopropanol, pipetting up and down gently (We invert the tubes to mix). (Do not vortex.)
2. Incubate the samples for 10 minutes at room temperature, then centrifuge at 14,000 x g for 30 minutes at 4°C.
3. The RNA precipitate forms a translucent gel-like pellet on the side and bottom of the tube.
D. RNA Wash
1. Remove all supernatant carefully (don’t lose the pellet).
2. Add 1 mL of 75% cold ethanol.
3. Mix by inversion. Do not vortex (The big difference here: Mix in the vortex! Like there is no tomorrow! LOL) Make sure that the pellet has released from the bottom. Centrifuge at 7,500 x g for 5 minutes at 4°C.
4. Remove the Ethanol carefully (don’t lose the pellet).
5. Repeat steps 1-4.
E. Resuspending the RNA
1. Remove supernatant with a pipet. Air-dry the RNA pellet by leaving the tubes open on the counter for approximately 15-30 minutes (Or in a heat block at 40 ºC until the pellet is dry).
2. Resuspend the RNA pellet in 50 µL of nuclease-free water. Mix by flicking incubating in a 40°C water bath for 10 minutes. The amount of RNAse/DNAse free water is variable according to the size of the pellet. You need to adjust. Check the concentration in a nanodrop and adjust as your need.
Best Regards,
Clei
We could really do with more detail re: "high-quality RNA".
What is the problem, precisely?
Yield?
260/280 (protein contam)?
260/230 (guanidium contam)?
Your protocol is ostensibly ok, though you don't say how _much_ tissue you add: too much tissue will saturate your TRIzol, and your yields and purity will be poor.
If in doubt, use less tissue, more TRIzol. You can have too much tissue, but you cannot have too much TRIzol.
Adding DNAse and then checking concentration will...give you really bad 260/280 ratios, because you've literally just added a load of protein to your sample, and then haven't removed it.
Add to this, if your 260/230 ratios are bad, the DNAse might not do anything (because adding an enzyme to a high concentration of chaotropic salts will...not end well for that enzyme).
If you provide more detail, we can probably be more helpful. RNA isolation is tricky, and sometimes it's tiny minor things that make all the difference.
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