| Description |
| The RT² qPCR-Grade miRNA Isolation Kit is
designed to enrich micro RNA from a total RNA preparation for real-time
RT-PCR based expression analysis. The kit, in combination with a
phenol/chloroform-based extraction method, prepares up to 10 µg of small
RNA, from cultured cells or animal tissues, that is free of genomic DNA,
large RNA, and other impurities. The special silica membrane spin column
technology, with appropriate ethanol concentrations, permits small RNA to
flow-through an initial column, while longer RNA and DNA bind. Upon
re-adjusting the ethanol concentration in the eluate, a second equivalent
column optimally retains the small RNA. Washing the second column removes
salts, metabolites, and other macromolecular cellular components. Low ionic
strength conditions finally elute pure small RNA ready for subsequent cDNA
template synthesis with the RTē miRNA First Strand Kit (MA-03) and miRNA
expression analysis optimized for the RTē miRNA qPCR System. The RTē qPCR-Grade
miRNA Isolation Kit provides an easy and efficient approach to acquire miRNA
of a greater purity and higher yield. The protocol takes only 60 minutes of
hands-on time, and the kit includes sufficient materials to purify 12 RNA
samples. |
| Materials Included /
Packing List |
Please check the kit components
immediately after you receive this package. SABiosciences is not responsible
for any missing items not reported within two (2) business days upon
receipt.
Storage Conditions: This kit is at ambient temperature. For long-term storage,
keep entire kit at room temperature.
Shelf Life: All reagents are stable for 6 months after receipt of the kit if
stored at the recommended temperature.
G6: Lysis and Binding Buffer
G17: Washing Buffer (Add 10 ml ethanol before use.)
RNase-free H2O
Spin Columns (24)
Collection Tubes (24)
Elution Tubes (12) |
| Brief Protocol: For Experienced
Users |
First-time Users should refer to the RT²
miRNA qPCR-Grade miRNA Isolation Kit User Manual.
- Sample Preparation:
Isolate total RNA from cells or tissues using the TRIzol protocol.
- Add 215 µL of 100% ethanol to a 400-µL sample. Mix well.
- Add sample to the center of a FIRST Spin Column.
Centrifuge for 30 s at 11,000 x g. Remove Spin Column from the Collection
Tube.
Discard FIRST Spin Column. SAVE Collection Tube AND Eluate.
- Add 750 µL of 100% ethanol to Eluate in Collection Tube. Mix well.
- Add 700 µL of mixture to the center of a SECOND Spin Column.
Centrifuge for 30 s at 11,000 x g. Remove Spin Column from Collection Tube.
Discard flow-through material. Place Spin Column back into Collection Tube.
- Add the remainder of the mixture to the center of the SAME SECOND Spin
Column.
Centrifuge for 30 s at 11,000 x g. Remove Spin Column from Collection Tube.
Discard flow-through material. Place Spin Column back into Collection Tube.
- Add 200 µL of Washing Buffer working solution (G17 plus ethanol) to Spin
Column.
Centrifuge for 30 s at 11,000 x g. Remove Spin Column from Collection Tube.
Discard flow-through material. Place Spin Column back into Collection Tube.
- Add 250 µL of 70% ethanol to each Spin Column.
Centrifuge for 3 min at 11,000 x g.
Transfer Spin Column from Collection Tube to Elution Tube. Discard
Collection Tube.
- Add 40 µL RNase-free H2O to each Spin Column.
Sit for 1 min at room temperature. Centrifuge for 1 min at 11,000 x g.
- Eluate contains enriched small RNA sample. Discard Spin Columns.
Store small RNA at -20 ºC for 2-4 days or at -80 ºC for up to six months.
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