PaxGene Blood RNA Kit Handbook - Plastisch chirurgen

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Appendix B: Quantification and Determination ofQuality of Total RNAQuantification of RNAThe concentration of RNA should be determined by measuring the absorbance at 260nm (A 260 ) in a spectrophotometer. To ensure significance, readings should be in thelinear range of the spectrophotometer. An absorbance of 1 unit at 260 nmcorresponds to 44 µg of RNA per ml (A 260 = 1 fi 44 µg/ml). This relation is valid onlyfor measurements in 10 mM Tris·Cl,* pH 7.5. Therefore, if it is necessary to dilute theRNA sample, this should be done in 10 mM Tris·Cl. As discussed below (see ”Purityof RNA”), the ratio between the absorbance values at 260 and 280 nm gives anestimate of RNA purity.When measuring RNA samples, be certain that cuvettes are RNase-free. Use the bufferin which the RNA is diluted to zero the spectrophotometer, and make sure to add thesame volume of Buffer BR5 as the volume of eluted RNA to be diluted.An example of the calculation involved in RNA quantification is shown below:Volume of RNA sample = 80 mlDilution = 10 ml of RNA sample + 140 ml 10 mM Tris·Cl, pH 7.5 (1/15 dilution)Measure absorbance of diluted sample in a cuvette (RNase-free).A 260 = 0.3Concentration of RNA sample = 44 x A 260 x dilution factor= 44 x 0.3 x 15= 198 mg/mlTotal yield= concentration x volume of sample in milliliters= 198 mg/ml x 0.08 ml= 15.8 mg RNA* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.For more information, consult the appropriate material safety data sheets (MSDSs), available from theproduct supplier.30PAXgene Blood RNA Kit Handbook 06/2005
Purity of RNAThe ratio of the readings at 260 nm and 280 nm (A 260 /A 280 ) provides an estimate ofthe purity of RNA with respect to contaminants that absorb in the UV, such as protein.However, the A 260 /A 280 ratio is influenced considerably by pH. Lower pH results in alower A 260 /A 280 ratio and reduced sensitivity to protein contamination.* For accuratevalues, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNAhas an A 260 /A 280 ratio of 1.8–2.2 in 10 mM Tris·Cl, pH 7.5. Always calibrate thespectrophotometer with the same solution.* Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on thespectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.PAXgene Blood RNA Kit Handbook 06/2005 31
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 12 and 13: RNA concentration and purificationT
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 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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