| Description |
| The ArrayGrade™ FFPE RNA Isolation Kit
is designed to isolate total RNA from formalin-fixed paraffin-embedded (FFPE)
blocks, sections or slides. The resulting RNA contains fewer cross-links
and serves as a better template for downstream applications such as
microarrays and real-time PCR with improved sensitivity. The protocol
includes deparaffinization, a treatment with a unique buffer system at an
elevated temperature to reverse cross-links more effectively than other
FFPE RNA isolation methods and a Proteinase K treatment to break up and
dissolve the tissue sample releasing the nucleic acids into solution. A
conventional total RNA isolation method then follows. Lysis buffer, with
its chaotropic components, stabilizes and prevents degradation of the RNA
and allows its optimal retention on the spin column. Washing Buffer
removes salts, metabolites, and macromolecular cellular components.
Finally, low ionic strength conditions elute pure RNA from the column
ready for gene expression analysis applications including labeled target
synthesis for microarrays and cDNA template synthesis for real-time PCR.
Need more information about GEArray products? please visit Microarray Home or send Email to Technical Support |
| 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.
We suggest that the kit contents be kept in their original container to
insure no components are misplaced.
|
TUBE
|
CONTENTS
|
| The following
components are shipped at ambient temperature. Unless otherwise noted,
store all components at room temperature. |
| G6 |
Lysis &
Binding Buffer
|
| G17 |
Washing Buffer
(Add 4 volumes of ethanol before use. See label for specific amount.)
|
| G26 |
RNase-free
10 mM Tris pH 8.0
|
| G50 |
Proteinase K
(Store at -20 ºC)
|
| G51 |
Proteinase K
Digestion Buffer
|
| G52 |
10X Un-Link-It
Buffer
|
| |
Snap-Cap Tubes,
Spin Columns, Collection Tubes (12 each) |
Shelf Life: All reagents are stable for 6 months after receipt
of the kit if stored at the recommended temperature. |
| Brief Protocol: For
Experienced Users |
First time users, please refer to the complete protocol in the User Manual.
- Follow the deparaffinization procedure previously used for your samples in
the provided Snap-Cap Tubes.(See the User Manual.)
- Add 100 µl of 1X Un-Link-It Buffer. Microwave the tubes in oven for 5
minutes at 50% power.
- Centrifuge at top speed (>13,000 x g), and carefully remove the
supernatant.
- Add 200 µl of Proteinase K digestion buffer. Wash the pellet by vortexing
briefly. Centrifuge at top speed (>13,000 x g) for 2 minutes. Carefully
remove the supernatant.
- Add 100 µl Proteinase K Digestion Buffer and 5 µl Proteinase K. Incubate
at 37 ºC for 1 to 2 hours. Flick tube every 15 to 20 minutes keeping tissue
dispersed. Stop incubation when solution clears and digestion is complete.
- Add 350 µl Lysis & Binding Buffer (G6). Mix by vortexing well.
Add 350 µl of 100% ethanol. Mix well by vortexing.
- Add sample to Spin Column. Centrifuge for 30 s at 8,000 x g. Discard the
flow-through material.
- Add 600 µl Washing Buffer working solution (G17 plus
ethanol) to Spin Column.
- Centrifuge for 30 s at 8,000 x g. Discard flow-through material.
- Add 200 µl Washing Buffer working solution (G17 plus
ethanol) to Spin Column.
- Centrifuge for 3 min at 11,000 x g to dry the column completely.
- Transfer Spin Column from Collection Tube into RNase-free 1.5-ml
microcentrifuge tube.
- Add 50 µl Buffer G26 to Spin Column. Incubate 1 min at room
temperature.
- Centrifuge for 1 min at 11,000 x g. Eluate contains total RNA sample.
|
