morgan@go2biotech.comGDP443-RNAprep Pure Hi-Blood Kit
RNAprep Pure Hi-Blood Kit
For purification of high-quality and stable total RNA from blood
RNAprep Pure Hi-Blood Kit
Cat.no. GDP443
Storage
The Buffer RLH added with β-mercaptoethanol can be kept at 2-8°C for one
month. DNase I and Buffer RDD should be stored at 2-8°C for 15 months.
Other reagents should be stored at room temperature (15-30°C) for 15
months.
Introduction
The kit can efficiently extract total RNA from fresh whole blood and blood
with various anticoagulants of different species. The silica matrix material
used in the adsorption column is a unique new material of TIANGEN,
which can adsorb RNA efficiently and specifically and can remove protein
impurities to the maximum extent. The extracted RNA can be used in
RT-PCR, Real Time RT-PCR, chip analysis, high-throughput sequencing,
Northern blot, Dot blot, Poly A screening, in vitrotranslation, RNase
protection analysis, molecular cloning , etc.
Please Pay Attention to The Following Aspect In Order to
Prevent RNase Pollution
RNase-free ddH2
O should be used to prepare the solution. (Add water
into a clean glass bottle, add DEPC to a final concentration of 0.1% (V/V),
mix well and then place overnight, sterilize with autoclave.)
Change new gloves frequently. Because the skin is often contaminated
with bacteria, it may contaminate RNase.
Use RNase-free plastic products and pipette tip to avoid cross
contamination.
RNA will not be degraded by RNase in Buffer RLH. However, RNase-free
plastic and glassware should be used in the further processing after
extraction. Bake glassware at 150°C for 4 h, or soak plastic ware in 0.5
M NaOH for 10 min, then thoroughly wash with water and autoclave to
remove RNase.
Important Notes Please read these notes before using this kit.
- Before operation, add β-mercaptoethanol to Buffer RLH to a final
concentration of 1%. (For example, add 10 μl β- mercaptoethanol to 1
ml RLH). It’s better to prepare the buffer when using. The prepared RLH
can be stored at 2-8°C for one month. The precipitate might formed
in Buffer RLH during storage.If there is precipitate, please use it after
heating and dissolving. - Add 96-100% ethanol to Buffer RW and Buffer RW1H before the first
time using. Please refer to the label on the bottle for the added volume. - Human blood or body fluid may have potential infectivity, so please pay
attention to take protective measures when processing human whole
blood. - The kit can process up to 1.5 ml of healthy human whole blood (the
leukocyte content in the whole blood of healthy people is up to 4000-7000 cells/μl blood). If the leukocyte content in the blood is high, the
amount of blood can be reduced proportionally, and the maximum
number of leukocyte that can be processed by the kit is 1×10
7
. - After leukocyte lysis, all the steps in this manual should be carried out
at room temperature (15-30°C), and please perform all steps as fast as
possible. - Cell lysate (in Buffer RLH) can be stored at -70°C When ready to use,
incubate it at 37°C for 10 minutes to ensure that all salts are dissolved,
then proceed to step 7 of the procedure. - This kit is not suitable for cryopreserved whole blood.
Preparation of DNase I storage solution
Dissolve DNase I dry powder (1500 U) in 550 μl RNase-Free ddH2
O, mix gently, and store at -30~-15°C after aliquoting (it can be stored for 9
months). (If DNase I is required, please enquire TIANGEN)
Note: DNase I storage solution thawed from -30~-15°C shall be stored at
2-8°C (can be stored for 6 weeks) and shall not be frozen again.
Protocol
- Dilution of Red Cell Lysis Buffer H: Select an appropriate volume of 10×
Red Cell Lysis Buffer H according to the volume of the treated blood
sample (for example, take 140 μl of 10× Red Cell Lysis Buffer H if the
volume of the processing blood sample is 200 μl), and dilute to 1× Red
Cell Lysis Buffer H with RNase-Free ddH2
O. - Add 5 times the volume of 1× Red Cell Lysis Buffer H to 1 volume of
human whole blood (proper clean tube should be self-provided).
Note: In order to obtain the best mixing effect, the volume of the
mixed solution of blood and 1× Red Cell Lysis Buffer H should not
exceed 3/4 of the tube volume. If the leukocyte content in blood is
high, the volume of blood used can be reduced proportionally, and the
volume of 1× Red Cell Lysis Buffer H used in step 6 should be adjusted
accordingly. - Incubate on ice for 10-15 min, and mix evenly for 2 times by vortex
during incubation.
Note: During the incubation, the solution will become translucent,
indicating red blood cells are lysed. If necessary, the incubation time
can be extended to 20 min. - Centrifuge at 4°C for 10 min at 2,100 rpm (~400 × g) to completely
remove the supernatant.
Note: Leukocyte may form globules after centrifugation. Make sure to
completely remove the supernatant. The presence of trace red blood
cells will make leukocyte globules appear red, and this phenomenon
will disappear in the subsequent washing step. - Add 1 × Red Cell Lysis Buffer H (the added volume of 1× Red Cell Lysis
Buffer H is 2 times of the total blood consumption in the step 1) to
leukocyte precipitation, and resuspend cells. - Centrifuge at 2,100 rpm (~400 × g) for 10 min at 4°C to completely
remove the supernatant.
Note: Incomplete removal of supernatant will affect lysis and the
subsequent binding of RNA to membrane, resulting in reduction of
final RNA yield. - Add Buffer RLH (please add β-mercaptoethanol before use)to
leukocyte precipitation. The specific dosage is as follows, vortex or mix
evenly with a pipette.
Note: If the blood is not the whole blood of a healthy person, the
volume of Buffer RLH should be determined according to the number
of leukocyte in the blood. At this time, the cells should be completely
lysed and the massive cell precipitation will disappear. - Transfer the solution to the RNase-Free Column CS (place the Column
CS in a collection tube) and centrifuge at 12,000 rpm (~ 13,400 × g) for 2
min, discard the Column CS and collect the filtrate.
Note: To avoid the formation of aerosol, please adjust the pipette to
≥ 750 ul to ensure that all the solutions are transferred to the filter
column at one time. If there are too many cells, the lysis solution will
be viscous and difficult to aspirate. - Add the same volume of 70% ethanol (usually 350 μl or 600 μl) to the
filtrate, mix well (precipitation may occur at this time), transfer the
obtained solution and precipitation into RNase-Free Column CR4 (place
the Column CR4 in a collection tube), centrifuge at 12,000 rpm (~13,400
× g) for 30-60 sec, discard the waste liquid in the collection tube, and
return Column CR4 to the collection tube.
Note: Use RNase-Free ddH2
O when preparing 70% ethanol. If there is
volume lost of filtrate, reduce the dosage of 70% ethanol accordingly.
If the solution volume is larger than the capacity of the CR4, so step 9
can be processed twice to collect all the solution. - If DNase I digestion is not carried out, 700 μl of Buffer RW1H can be
directly added to Column CR4 (ensure that ethanol has been added
before use). Centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec,
discard waste liquid in the collection tube, and directly proceeded to
step 14.DNase I digestion: Add 350 μl of Buffer RW1H to Column CR4,centrifuge at 12,000 rpm (~13,400 x g) for 30-60 sec, discard wasteliquid in the collection tube and put the Column CR4 back into the collection tube. - Preparation of DNase I working solution: Transfer 10 μl DNase I
storage solution to a new RNase-Free centrifuge tube, add 70 μl RDD
solution, and mix gently. - Add 80 μl of DNase I working solution to the center of Column CR4
and place at room temperature for 15 min. - Add 350 μl Buffer RW1H to Column CR4, centrifuge at 12,000 rpm
(~13,400 × g) for 30-60 sec, discard the waste liquid in the collection
tube, and return Column CR4 to the collection tube. - Add 500 μl of Buffer RW to Column CR4 (ensure that ethanol has
been added before use). Incubate at room temperature for 2 min,
centrifuge at 12,000 rpm (~13,400 × g) for 30-60 sec, discard the
waste liquid in the collection tube, and put Column CR4 back into
the collection tube. - Repeat step14.
- Centrifuge at 12,000 rpm (~13,400 × g) for 2 min and discard the
waste liquid. Place the Column CR4 at room temperature for several
minutes to completely dry the residual washing buffer in the
adsorption material.
Note: After centrifugation, leave Column CR4 at room temperature
for several minutes to fully dry the membrane. If there is Buffer
RW residue, it may affect subsequent reverse transcription,
fluorescence quantification and other experiments. - Transfer the adsorption column CR4 into a new RNase-Free
centrifuge tube, add 30-50 μl RNase-Free ddH2
O and leave at room
temperature for 2 min, centrifuge at 12,000 rpm (~13,400 × g) for 2
min to obtain the solution.
Note: The volume of elution buffer should not be less than 30
μl. Too small a volume will affect the recovery of efficiency. RNA
solution should be stored at -70°C.
GDP443-RNAprep Pure Hi-Blood Kit
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