morgan@go2biotech.comGDP503-miRcute SerumPlasma miRNA Isolation Kit
miRcute Serum/Plasma miRNA Isolation Kit
High-efficiency miRNA extraction kit specifically targeting serum/plasma samples
miRcute Serum/Plasma miRNA Isolation Kit
(Spin Column)
Cat. no. GDP503
Storage
Buffer MZA should be stored at 2-8°C protected from light for 15 months.
The miRcute Serum/plasma miRNA Isolation Kit should be stored dry at
room temperature(15-30°C) for 15 months.
Introduction
The miRcute Serum/Plasma miRNA Isolation Kit is designed for isolation
and purification of miRNA from serum/plasma. The optimized lysis buffer
in the kit has super lysis ability and isolation sensitivity. The kit utilizes
a silica-based system to adsorb RNA, especially small RNA (<200 nt).
miRNA with high purity and quality with no contamination of DNA and
protein could be obtained within 1 hour. The purified miRNA can be used
in Northern Blot, Dot Blot, Poly A screening, in vitro translation, RNase
protection analysis and molecular cloning, etc.
Precautions Please read these precautions before use
To avoid RNase contamination, please note that:
- Change gloves regularly, for bacteria on the skin can result in RNase
contamination. - Use RNase-Free plastic and tips to avoid cross contamination.
- RNA can be protected in Buffer MZA. The following steps after lysis
should be performed in RNase-Free plastic or glassware. To wipe off
RNase, the glassware can be heated at 150°C for 4 hours, and plastic can
be dipped in 0.5 M NaOH for 10 min, and washed by RNase-Free ddH2O
thoroughly, then send for autoclaving. - Use RNase-Free ddH2O to prepare the solutions. (Add DEPC to 0.1%
final concentration in ddH2O. Shake to mix, and leave overnight at room
temperature, then autoclave for 15 min).
Protocol
Ensure ethanol (96-100%) has been added to Buffer RW and Buffer MRD
with appropriate volume as indicated on the bottle and shake thoroughly.
- Preparation of samples: Add 900 μl Buffer MZA to 200 μl serum or
plasma, vortex 30 s to mix thoroughly, and then invert up and down
to mix. If the External Control (Cat.no. GCR100-01) is applied, please
add in the External Control (for 1 μM, add in 1 μl) after the complete
homogenization and before the inverting mixing.
Note: The starting volume of sample should be no more than 200
µl, otherwise RNA yield and purity will be low. The volume of Buffer
MZA must strictly in accordance with the volume in the instructions.
If the amount of Buffer MZA is small, the phase separation will not be
complete, which will cause low yield and purity of RNA. - Place the tube containing the homogenate on the benchtop at room
temperature (15-30°C) for 5 min to separate nucleic acids and protein. - Add 200 μl chloroform to the supernatant and cap it securely. Shake the
tube vigorously for 15 s, incubate the tube containing the homogenate
on the bench top at room temperature for 5 min. - Centrifuge for 15 min at 12,000 rpm (~13,400 × g) at 4°C. After centrifugation,
the sample separates into 3 phases: An upper, colorless, aqueous phase
containing RNA; A white interphase; And a lower yellow organic phase.
Transfer the aqueous phase to a new tube and proceed to the next step. - Add 2× the volume of ethanol (96-100%) to the aqueous phase solution
(e.g. add 1 ml ethanol (96-100%) to 500 μl aqueous phase solution) and mix
thoroughly by pipetting up and down several times (precipitate may form
after addition of ethanol, but this will not affect the procedure). Transfer
the obtained liquidand any precipitate that may have formed into a RNaseFree Column miRelute, incubate at room temperature for 2 min, then
centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard the flow through
after centrifugation, and keep the Column miRelute. - Add 700 μl Buffer MRD (ensure that ethanol (96-100%) has been
added) to the Column miRelute . Close the lid gently and incubate 2 min
at room temperature. Then centrifuge for 30 s at 12,000 rpm (~13,400 ×
g) to wash the column. Discard the flow through. - Add 500 μl Buffer RW (ensure that ethanol (96-100%) has been added)
to the Column miRelute . Close the lid gently and incubate 2 min at
room temperature. Then centrifuge for 30 s at 12,000 rpm (~13,400 × g)
to wash the column. Discard the flow-through. - Repeat step 7.
- Centrifuge at 12,000 rpm (~13,400 × g) for 2 min at room temperature,
and discard the 2 ml collection tube.
Note: This step is to eliminate any possible carry-over of Buffer RW.
After centrifugation. place the Column miRelute at clean bench for a
while to completely dry the column membrane. Residual Buffer RW
will have negative influence on following RT experiment. - Transfer the Column miRelute to a new 1.5 ml RNase-Free Centrifuge
Tube, add 15-30 μl RNase-Free ddH2
O directly onto the miRelute column
membrane and incubate 2 min at room temperature. Close the lid gently and
centrifuge for 2 min at 12,000 rpm (~13,400 × g) to elute the RNA.
Note: The volume of elution buffer should not be less than 15 μl. Small
elution volume may have a negative effect on RNA yield. Please store
RNA solution at -70°C. To obtain a higher total RNA concentration,
please repeat step 10; or increase sample volume and Buffer MZA
and chloroform volume in proportion.
GDP503-miRcute SerumPlasma miRNA Isolation Kit
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This product is for scientific research use only. Do not use in medicine, clinical treatment, food or cosmetics.
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E-mail: maggie@go2biotech.com / morgan@go2biotech.com
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