Blood RNA rapid extraction steps
Steps:
prompt:
(1) Add the specified amount of absolute ethanol to the rinse RW bottle and the 70% ethanol bottle before using for the first time!
(2) Dilute 10X red blood cell lysate RLB to 1X with DEPC treated water before use.
(3) Add β-mercaptoethanol to the lysate RLT before the operation to a final concentration of 1%, such as 10 μl of β-mercaptoethanol in 1 ml of RLT. This lysate is preferably used now. With a good RLT 4 ° C can be placed for one month.
1. Add 1 volume (<1.5 ml) to the appropriate size of the RNase free centrifuge tube, add various anticoagulant fresh blood (after mixing) and 3 volumes of red blood cell lysate RLB, mix by inversion, and flick the tube. Wall, make sure to mix.
2. Leave at room temperature for 10 minutes (during the period of time, you should reverse the flick and mix several times to help lyse the red blood cells).
If the RNA is severely degraded, it can be cleaved on ice, but the time can be longer to fully lyse.
3. Centrifuge at 12,000 rpm for 20 seconds, discard the red supernatant, and carefully discard the supernatant as much as possible (be careful not to aspirate the cell mass at the bottom of the tube), leaving a complete tube bottom cell mass.
After centrifugation, white leukocyte clusters should be seen at the bottom of the tube. There may also be some red blood cell debris and white blood cell clusters together. However, if most of the red cell clusters are seen, the red blood cell lysis is not sufficient, and red blood cell lysis should be added. Repeat steps 2 and 3 after resuspending the cell pellet.
The supernatant is as much as possible, and excessive residue will dilute the lysate, resulting in abnormal cleavage and a decrease in yield purity.
4. Vortex or flick the wall to completely loosen and resuspend the leukocyte pellet. Add 350 μl (<0.5 ml whole blood) or 600 μl (0.5-1.5 ml whole blood) lysate RLT, mix by blowing and shake vigorously for 20 seconds by hand. , fully lysed. The number of white blood cells in the patient's blood sample may increase significantly, and the amount of treatment should be appropriately reduced. Or add RTL in a ratio of 350 μl (<2x106 white blood cells) or 600 μl (2 x 106-1 x 107 white blood cells).
5. Pump the lysate 5-10 times with a disposable 1 ml (with a 0.9 mm needle) syringe with a blunt needle or until a satisfactory homogenization result (or motor homogenization for 30 seconds) is performed to cut the DNA and reduce the viscosity. And increase production.
6. Accurately estimate the lysate volume, add an equal volume of 70% ethanol (please check if you have added absolute ethanol!), precipitation may occur at this time, but does not affect the extraction process, immediately mix and mix, do not centrifuge.
7. Immediately add the mixture (less than 700 μl each time, can be added in two portions) to an adsorption column RA (the adsorption column is placed in a collection tube) and centrifuge at 13,000 rpm for 60 seconds to discard the waste liquid.
8. Add 700 μl of deproteinized solution RW1, leave it at room temperature for 30 seconds, centrifuge at 12,000 rpm for 30 seconds, and discard the waste.
If the DNA residue is obvious, it can be placed at room temperature for 5 minutes after adding RW1 and then centrifuged.
9. Add 500 μl of rinse RW (check to see if absolute ethanol has been added!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste. Add 500 μl of rinse RW and repeat.
10. Place the adsorption column RA back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove the rinse solution as much as possible to prevent residual ethanol in the rinse solution from inhibiting the downstream reaction.
11. Remove the adsorption column RA and place it in an RNase free centrifuge tube. Add 30-50μl RNase freewater in the middle of the adsorption membrane according to the expected RNA yield (previously heated in 70-80 °C water bath), place at room temperature. Centrifuge at 12,000 rpm for 1 minute.
12. If you are taking whole blood >0.5 ml or >2x106 white blood cells, add 30-50μl RNase free water to repeat step 11 and combine the two eluates, or use the first eluent to add back to the adsorption column and repeat the steps ( If the RNA concentration is high).
The concentration of the RNA eluate eluted twice was high, and the RNA yield of the eluate was 15–30% higher than that of the former, but the concentration was low, and the user selected it as needed.
prompt:
(1) Add the specified amount of absolute ethanol to the rinse RW bottle and the 70% ethanol bottle before using for the first time!
(2) Dilute 10X red blood cell lysate RLB to 1X with DEPC treated water before use.
(3) Add β-mercaptoethanol to the lysate RLT before the operation to a final concentration of 1%, such as 10 μl of β-mercaptoethanol in 1 ml of RLT. This lysate is preferably used now. With a good RLT 4 ° C can be placed for one month.
1. Add 1 volume (<1.5 ml) to the appropriate size of the RNase free centrifuge tube, add various anticoagulant fresh blood (after mixing) and 3 volumes of red blood cell lysate RLB, mix by inversion, and flick the tube. Wall, make sure to mix.
2. Leave at room temperature for 10 minutes (during the period of time, you should reverse the flick and mix several times to help lyse the red blood cells).
If the RNA is severely degraded, it can be cleaved on ice, but the time can be longer to fully lyse.
3. Centrifuge at 12,000 rpm for 20 seconds, discard the red supernatant, and carefully discard the supernatant as much as possible (be careful not to aspirate the cell mass at the bottom of the tube), leaving a complete tube bottom cell mass.
After centrifugation, white leukocyte clusters should be seen at the bottom of the tube. There may also be some red blood cell debris and white blood cell clusters together. However, if most of the red cell clusters are seen, the red blood cell lysis is not sufficient, and red blood cell lysis should be added. Repeat steps 2 and 3 after resuspending the cell pellet.
The supernatant is as much as possible, and excessive residue will dilute the lysate, resulting in abnormal cleavage and a decrease in yield purity.
4. Vortex or flick the wall to completely loosen and resuspend the leukocyte pellet. Add 350 μl (<0.5 ml whole blood) or 600 μl (0.5-1.5 ml whole blood) lysate RLT, mix by blowing and shake vigorously for 20 seconds by hand. , fully lysed. The number of white blood cells in the patient's blood sample may increase significantly, and the amount of treatment should be appropriately reduced. Or add RTL in a ratio of 350 μl (<2x106 white blood cells) or 600 μl (2 x 106-1 x 107 white blood cells).
5. Pump the lysate 5-10 times with a disposable 1 ml (with a 0.9 mm needle) syringe with a blunt needle or until a satisfactory homogenization result (or motor homogenization for 30 seconds) is performed to cut the DNA and reduce the viscosity. And increase production.
6. Accurately estimate the lysate volume, add an equal volume of 70% ethanol (please check if you have added absolute ethanol!), precipitation may occur at this time, but does not affect the extraction process, immediately mix and mix, do not centrifuge.
7. Immediately add the mixture (less than 700 μl each time, can be added in two portions) to an adsorption column RA (the adsorption column is placed in a collection tube) and centrifuge at 13,000 rpm for 60 seconds to discard the waste liquid.
8. Add 700 μl of deproteinized solution RW1, leave it at room temperature for 30 seconds, centrifuge at 12,000 rpm for 30 seconds, and discard the waste.
If the DNA residue is obvious, it can be placed at room temperature for 5 minutes after adding RW1 and then centrifuged.
9. Add 500 μl of rinse RW (check to see if absolute ethanol has been added!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste. Add 500 μl of rinse RW and repeat.
10. Place the adsorption column RA back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove the rinse solution as much as possible to prevent residual ethanol in the rinse solution from inhibiting the downstream reaction.
11. Remove the adsorption column RA and place it in an RNase free centrifuge tube. Add 30-50μl RNase freewater in the middle of the adsorption membrane according to the expected RNA yield (previously heated in 70-80 °C water bath), place at room temperature. Centrifuge at 12,000 rpm for 1 minute.
12. If you are taking whole blood >0.5 ml or >2x106 white blood cells, add 30-50μl RNase free water to repeat step 11 and combine the two eluates, or use the first eluent to add back to the adsorption column and repeat the steps ( If the RNA concentration is high).
The concentration of the RNA eluate eluted twice was high, and the RNA yield of the eluate was 15–30% higher than that of the former, but the concentration was low, and the user selected it as needed.
Henan Aklly Filter Engineering Co., Ltd , https://www.akllyfilter.com