Whole blood RNA extraction
Whole blood RNA extraction
Whole blood RNA extraction can be used (1) to study the mechanism of RNA interference, etc.; (2) as a template for reverse transcription; and (3) for other studies in molecular biology.
Operation method
Whole Blood RNA Extraction
Principle
Erythrocyte lysate selectively lyses erythrocytes, then the unique lysate/β-mercaptoethanol rapidly lyses leukocytes and inactivates the cellular RNAase, and then after adjusting the binding conditions with ethanol, the RNA is selectively adsorbed onto the silica matrix membrane inside the centrifugal column in the high salt state, and then through a series of rapid rinsing-centrifugation steps, the deproteinizing solution and the rinsing solution remove the cellular metabolites, proteins, and other impurities, and then the low-salt RNase free H20 elutes pure RNA from the silica matrix membrane. Finally, pure RNA is eluted from the silica membrane with low salt RNase free H20.
Materials and Instruments
Fresh Blood Move i. Add 1 volume (<1.5 ml) of fresh blood with various anticoagulants (after inverted mixing) and 3 volumes of red blood cell lysate RLB to an appropriately sized RNase free centrifuge tube and mix inverted, flicking the walls of the tube to ensure mixing. Caveat 1. Add the indicated amount of anhydrous ethanol to the Rinse Solution RW bottle and 70% ethanol bottle before first use. 2. Dilute 10X Red Blood Cell Lysate RLB to 1X with DEPC-treated water before use. 3. Add β-mercaptoethanol to the lysate RLT to a final concentration of 1% prior to operation, e.g., 10 ul β-mercaptoethanol to 1 ml RLT. This lysate is best prepared on the spot. The prepared RLT can be left at 4°C for one month. Common Problems For more product details, please visit Aladdin Scientific website.
Anticoagulants Erythrocyte Lysate RLB Deproteinizing Solution RW1 Ethanol Rinsing Solution RW
Ice maker Centrifuge Centrifuge tube Pipette gun Pipettes Needles Syringes Water baths
Allow to stand at room temperature for 10 minutes (during which time the tube should be inverted and flicked several times to aid in lysing the erythrocytes).
If the RNA is heavily degraded, it can be lysed on ice, but for a longer period of time to fully lysed.
III. Centrifuge at 12,000 rpm for 20 seconds, discard the red supernatant, and carefully aspirate as much of the supernatant as possible (being careful not to aspirate the cell mass at the bottom of the tube), leaving a complete mass of leukocytes at the bottom of the tube.
After centrifugation, you should see white leukocyte clusters at the bottom of the tube, and there may be some red cell debris with the leukocyte clusters, but if you see mostly red cell clusters, the erythrocyte lysis is very insufficient, and you should add more erythrocyte lysate to resuspend the clusters and repeat steps two and three.
The supernatant should be aspirated and discarded as much as possible, as too much residue will dilute the lysate, resulting in abnormal lysate binding and lower yield purity.
Vortex or flick the wall of the tube to completely loosen and resuspend the leukocyte precipitate, add 350 ul (<0.5 ml of whole blood) or 600 ul (0.5-1.5 ml of whole blood) of lysate RLT, blow and mix well and then shake vigorously by hand for 20 seconds to fully lysate. The patient's blood sample may have a substantially higher leukocyte count and should be processed in an appropriately reduced amount. Alternatively, add RTL at a ratio of 350 ul ( <2x106 leukocytes) or 600 ul ( 2x106-1x107 leukocytes).
v. Drawing lysates with a disposable 1 ml (with 0.9 mm needle) syringe with a blunt tip 5-10 times or until satisfactory homogenization results are obtained (or motorized homogenization for 30 seconds) shears the DNA, reduces the viscosity and increases the yield.
Sixth, estimate the volume of lysate more accurately and add an equal volume of 70% ethanol (please check that anhydrous ethanol has been added first!). , precipitation may appear at this time, but it does not affect the extraction process, blow and mix immediately, do not centrifuge.
VII. Immediately add the mixture (less than 700 ul each time, more can be added in two times) to an adsorption column RA, (adsorption column into the collection tube) centrifugation at 13 000 rpm for 60 seconds, discard the waste liquid.
Add 700 ul of deproteinizing solution RW1, leave at room temperature for 30 seconds, centrifuge at 12,000 rpm for 30 seconds and discard the waste solution.
If DNA residues are evident, leave at room temperature for 5 minutes after addition of RW1 before centrifugation.
Add 500 ul of Rinse Solution RW (please check if anhydrous ethanol has been added first), centrifuge at 12 000 rpm for 30 seconds, discard the waste solution. Add 500 ul Rinse Solution RW and repeat.
Place the column RA back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove as much of the rinse solution as possible so that ethanol residues in the rinse solution do not inhibit downstream reactions.
XI. Remove the adsorbent column RA, put it into an RNase free centrifuge tube, add 30-50 ul of RNase freewater to the middle part of the adsorbent membrane according to the expected RNA yield (it is better to heat it up in a 70-80℃ water bath beforehand), leave it at room temperature for 1 minute, and then centrifuge it at 12,000 rpm for 1 minute.
If whole blood >0.5 ml or >2x106 leukocytes are extracted, add 30-50 ul of RNase free water and repeat step 11, combining the two eluents, or use the first eluent and add it back to the column and repeat the step again (if a high RNA concentration is required).
The RNA eluent eluted twice has a high concentration, and the RNA yield of combining the eluents in two separate elutions is 15-30% higher than the former, but the concentration is lower, so the user chooses according to the need.