RT² qPCR-Grade RNA Isolation Kit
Catalog Number Format
PA-001 Enough reagents for 12 total RNA preparations
 
Description
The RT² qPCR-Grade RNA Isolation Kit is designed to extract total RNA that is free of genomic DNA and other impurities from typical quantities of biological source material. This kit prepares up to 100 µg of total RNA directly from transformed animal cell lines or is used in combination with a phenol/chloroform-based extraction method to isolate high quality total RNA from animal tissues. The kit also includes a step that very effectively eliminates genomic DNA contamination from RNA samples during the isolation procedure. The special silica membrane Spin Column technology used in the kit makes the procedure fast and easy to perform with less than 30 minutes of hands-on time at room temperature. The lysis buffer, with its chaotropic components, stabilizes RNA, prevents its degradation by inhibiting RNase activity, and optimizes the conditions for its retention on the spin column. An on-column treatment with an RNase-free DNase enzyme specifically digests genomic DNA into material unable to bind the column, while leaving the retained RNA perfectly intact. The Washing Buffers remove the degraded genomic DNA, salts, metabolites, and macromolecular cellular components. Low ionic strength conditions elute pure RNA from the column. The RNA is then ready for subsequent cDNA template synthesis using the RT² First Strand Kit.
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.
TUBES AND CONTENTS
Box 1
RNase-free DNase (rDNase)
DNase Reaction Buffer
Box 2
Lysis and Binding Buffer (G6)
Desalting Buffer (G15)
Pre-wash Buffer (G16)
Washing Buffer (G17: Add 10 ml ethanol before use.)
RNase-free H2O
Spin Columns (12)
Collection Tubes (36)
Filter Columns (12)
Elution Tubes (12)
We suggest that the kit contents be kept in their original container to insure that no components are misplaced.
Storage Conditions:  
BOX 1 is shipped on blue ice packs or dry ice and should be stored at -20 ºC upon receipt.
BOX 2 is shipped at ambient temperature and should be stored at room temperature.
Shelf Life: All reagents are stable for 6 months after receipt of the kit if stored at the recommended temperature.
Brief Protocol
First time users, please refer to the complete protocol in the User Manual.
  1. Sample Preparation
    • Cell Pellets:
      Add 350 µL of Lysis and Binding Buffer (G6) to cell pellet in a 1.5-ml RNase-free tube.
      Add mixture to Filter Column in Collection Tube, and centrifuge for 1 min at 11,000 x g.
      Save eluate. Discard the Filter Column.
    • Tissue Samples:
      Isolate RNA using the Invitrogen TRIzol® protocol first, making sure that the TRIzol® reagent volume is at least ten-times the tissue sample volume.
      Add 350 µL of Lysis and Binding Buffer (G6) to RNA re-suspended after the ethanol precipitation step in no more than 50 µL of RNase-free H2O.
    • Previously Isolated yet still crude RNA
      Add 350 µL of Lysis and Binding Buffer (G6) to RNA in up to 50 µL of RNase-free H2O.
      Mix by vortexing well.
  2. Add 350 µL of ethanol (70%) to the sample and mix by pipetting up-and-down five (5) times or by vortexing gently.
  3. Load the entire volume (up to 750 µL) onto Spin Column. Centrifuge for 30 s at 11,000 x g.
    Discard flow-through material. Place Spin Column back into Collection Tube.
  4. Add 350 µL of Desalting Buffer (G15) onto spin column; centrifuge for 60 s at 11,000 x g.
    Discard flow-through material. Transfer Spin Column to NEW Collection Tube.
  5. Mix 10 µL of RNase-free DNase (rDNase) and 90 µL of DNAse Reaction Buffer.
    Add entire volume to Spin Column. Incubate at room temp for 15 min.
  6. Add 200 µL of Pre-Wash Buffer (G16) onto spin column; centrifuge for 30 s at 11,000 x g.
    Discard flow-through material. Transfer Spin Column to NEW Collection Tube.
  7. Add 600 µL of Washing Buffer (G17 + ethanol) onto spin column; centrifuge for 30 s at 11,000 x g. Discard flow-through material. Place Spin Column back into Collection Tube.
  8. Add 250 µL of Washing Buffer (G17 + ethanol) onto spin column; centrifuge for 3 min at 11,000 x g. 
    Discard flow-through material. Transfer Spin Column to Elution Tube.
  9. Add 50 µL of RNase-free H2O to the spin column. Incubate at room temperature for 1 min.
    Centrifuge for 1 min at 11,000 x g.
  10. Store RNA at -20 ºC for two to four days or at -80 ºC for up to six months.
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