morgan@go2biotech.comGDP502-miRNAprep Pure FFPE Kit
miRNAprep Pure FFPE Kit
Highly efficient extraction of RNA>18 nt from paraffin sections
miRNAprep Pure FFPE Kit
(Spin Column)
Cat.no. GDP502
Storage
DNase I, Buffer RDD and RNase-Free ddH2O (tube) 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 adopts a special buffer system and optimized protocol, which
is especially designed for miRNA extraction in formalin-fixed paraffinembedded tissue sections (hereinafter referred to as FFPE). The kit has the
characteristics of high specificity, fast experiment, high quality of extractedmiRNA, etc.
RNA extracted by the kit can be applied to downstream tests such as RT-PCR.
Precautions Please read these precautions before use
- 96%-100% ethanol should be added to Buffer RW before using for the
first time. Please refer to the label on the bottle for the added amount. - 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 (can be stored for 9
months).
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.
Starting material
- Standard formalin-fixed paraffin embedding procedures often cause
fragmentation of nucleic acids. To minimize the possibility of RNA
fragmentation, sample processing should be carried out according to
the following operating steps:
● The tissue should be immersed in 4%-10% formalin solution as soon
as possible after excision.
● The fixing time is preferably 14-24 h (Too long fixing time will lead
to more serious RNA fragmentation, which is not conducive to
downstream tests);
● The sample must be completely dehydrated before being coated. - Use fresh FFPE tissue slices.The slice thickness should not exceed 10
μm. Too thick slices may cause low RNA yield. The number of slices used
for each preparation should not exceed 8 slices, and the surface area
should not exceed 250 mm2. - If no information is available on the initial sample, It is suggested that the
number of FFPE slices used for initial preparation should not exceed 2 slices,
and then the number of slices used for next preparation can be adjusted
according to the experimental results, but should not exceed 8 slices.
Protocol
Be sure to add ethanol to Buffer RW as indicated on the bottle and shake
thoroughly.
- Cut the FFPE sample into 5-10 μm thick sheets.
Note: If the sample surface is exposed to air, discard the first 2-3
pieces. - Quickly place 2-8 slices into a 1.5 ml RNase-free centrifuge tube, add 1
ml xylene, and vortex for 10 sec. - Centrifuge at room temperature (15-30°C) for 2 min at 12,000 rpm
(~13,400×g). - Remove the supernatant with the pipette tip and be careful not to
disturb the precipitate. - Add 1 ml of 96-100% ethanol into the precipitate, and mix well by
vortex. - Centrifuge at room temperature for 2 min at 12,000 rpm (~13,400×g).
- Remove the supernatant with a pipette and be careful not to disturb
the precipitate. (Use a new pipette tip to carefully aspirate the residual
ethanol). - Open the tube cover and leave at room temperature or 37°C for 10 min
until the residual ethanol volatilizes completely.
Note: It is very important to completely remove the residual ethanol,
or it will affect the extraction of RNA. - Add 150 μl of Buffer RF and 10 μl of Proteinase K to the precipitate and
thoroughly mixed by vortex. - Incubate at 55°C for 15 min and then at 80°C for 15 min.
- Place on ice for 3 min, centrifuge at 12,000 rpm (~13,400×g) for 15
min, and transfer the supernatant to a new 2 ml RNase-Free centrifuge
tube. - Add 16 μl of Buffer RDD and 10 μl of DNase I solution, and mix them by
turing up and down. Leave at room temperature for 15 min. Centrifuge
briefly to collect droplets on the wall and lid of the tube. - Add 320 μl of Buffer RB and mix well by vortex.
- Add 1120 μl of 96%-100% ethanol and mix well by vortex (precipitation
may occur). - Transfer 700 μl of solution and precipitate into RNase-Free Column
CR3 (place the CR3 in the collection tube), centrifuge at 12,000 rpm
(~13,400×g) for 1 min, discard the waste liquid in the collection tube,
and return the Column CR3 to the collection tube. - Repeat step 15 until all the solution and precipitate completely pass
through the Column CR3, discard the waste liquid, and put the Column
CR3 back into the collection tube. - Add 500 μl of Buffer RW (ensure that ethanol has been added before
use)to Column CR3, let it stand at room temperature for 2 min,
centrifuge at 12,000 rpm (~ 13,400 x g) for 30-60 sec, discard the waste
liquid, and return Column CR3 to the collection tube. - Repeat step 17.
- Centrifuge at room temperature for 2 min at 12,000 rpm (~13,400×g)
and discard the waste liquid. Place the Column CR3 at room
temperature for several minutes to completely dry the residual
washing buffer in the Column CR3.
Note: The purpose of this step is to remove the residual Buffer RW in
Column CR3. The residual Buffer RW may affect subsequent RT and
other experiments. - Transfer the Column CR3 into a new RNase-Free centrifuge tube,
add 30-100 μl RNase-Free ddH2
O into the middle of the adsorption
membrane. Incubate at room temperature for 2 min, then centrifuge
for 2 min at 12,000 rpm (~13,400×g).
Note: The volume of elution buffer should not be less than 30 μl. Too
small volume will affect the recovery efficiency. RNA solution should
be stored at -70°C.
GDP502-miRNAprep Pure FFPE Kit
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E-mail: maggie@go2biotech.com / morgan@go2biotech.com
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