PaxGene Blood RNA Kit Handbook - Plastisch chirurgen

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RNA concentration and purificationThe PAXgene Blood RNA Kit is for the purification of total RNA from 2.5 ml humanwhole blood collected in a PAXgene Blood RNA Tube. The procedure is simple (seeflowchart, page 13). Purification begins with a centrifugation step to pellet nucleic acidsin the PAXgene Blood RNA Tube. The pellet is washed and resuspended, and incubatedin optimized buffers together with proteinase K to bring about protein digestion. Anadditional centrifugation through the PAXgene Shredder spin column is carried out tohomogenize the cell lysate and remove residual cell debris, and the supernatant of theflow-through fraction is transferred to a fresh microcentrifuge tube. Ethanol is added toadjust binding conditions, and the lysate is applied to a PAXgene RNA spin column.During a brief centrifugation, RNA is selectively bound to the PAXgene silica membraneas contaminants pass through. Remaining contaminants are removed in several efficientwash steps. Between the first and second wash steps, the membrane is treated withDNase I to remove bound DNA. After the wash steps, RNA is eluted in Buffer BR5.Total RNA purified using the PAXgene Blood RNA System is highly pure, with A 260 /A 280values between 1.8 and 2.2 and ≤1.0% (w/w) genomic DNA. At least 95% of samplesshow no inhibition in RT-PCR, when using up to 30% of the eluate.Average sample preparation time (based on data from 12 sample preps) isapproximately 90 minutes, with only 40 minutes of hands-on time. RNA yields from2.5 ml healthy human whole blood are ≥3 µg for ≥95% of the samples processed. Sinceyields are highly donor-dependent, individual yields may vary. For individual donors,the PAXgene Blood RNA System provides highly reproducible and repeatable yields(Figures 3 and 4, pages 14 and 15) and reproducible and repeatable RT-PCR(Figures 5 and 6, pages 18 and 19), making it highly robust for clinical diagnostic tests.12PAXgene Blood RNA Kit Handbook 06/2005
The PAXgene Blood RNA ProcedureBloodIncubateTransfer to PAXgeneShredder Spin ColumnMixTransfer supernatant of flow-throughto microcentrifuge tubeAdd ethanolLoad on PAXgene RNASpin ColumnWash pelletBind total RNAWashDigest DNAResuspendTransfer tomicrocentrifugetubeAdd proteinase Kand binding bufferWashEluteHeat to 65°CReady-to-use RNAPAXgene Blood RNA Kit Handbook 06/2005 13
Page 1 and 2: Σ50 (cat. no. 762164)PAXgeneBlood
Page 3 and 4: ContentsExplanation of Symbols 4Kit
Page 6 and 7: Intended UseThe PAXgene Blood RNA K
Page 8 and 9: Buffer BR3Contains ethanol: flammab
Page 10 and 11: RNA Stability in Blood Samples at 1
Page 14 and 15: A2420Reproducible and Repeatable RN
Page 16 and 17: Table 1A. Reproducibility within Ea
Page 18 and 19: Reproducibility of RT-PCR — betwe
Page 20 and 21: Table 2. Summary of RT-PCR Data fro
Page 22 and 23: Protocol: Purification of Total RNA
Page 24 and 25: ProtocolProcedure1. Centrifuge the
Page 26 and 27: Protocol15. Pipet 500 µl Buffer BR
Page 28 and 29: Comments and suggestionsLow A 260 /
Page 30 and 31: Appendix B: Quantification and Dete
Page 32 and 33: Appendix C: Handling PAXgene Blood
Page 34 and 35: PreAnalytiX products are distribute
Page 36: 1028434 06/2005

PaxGene Blood RNA Kit Handbook - Plastisch chirurgen

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