RNA Cell HTS 96 Kits

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Principle and procedureThe InviTrap ® RNA Cell HTS 96 Kit procedure comprises following steps:1. lysis of cells2. selective binding of the genomic DNA to specific carrier and separation of contaminating DNAon the surface of the DNA Binding Plate3. remaining sample transferring into the wells of the RNA binding plate, followed by theadjustment of the binding conditions4. binding of the total RNA to the membrane5. washing of the membrane and elimination of contaminants and ethanol6. elution of high pure total RNAAfter lysis the DNA binds onto membrane of the DNA binding plate, after contaminations andenzyme inhibitors are efficiently removed during the following two wash steps and highly purifiedRNA is eluted in Elution Buffer or water.This manual contains 3 protocols (page 13-17).Sampling and storage of starting material:Best results are obtained using freshly extracted cells. As long as samples aren’t shock frosted orare inserted in RNase inhibitors or denaturing reagents, RNA is not secured. Therefore it isessential, that cells are immediately flash frozen after cell harvest and are stored at –80°C. RNApurification should be processed as soon as possible. Cells also can be stored in Lysis Solution Sat –80°C after cell lysis.RNA from deep frozen samples is stable for months.LysisAfter removing of the culture medium, cells are directly charged with DTT-containing LysisSolution S and thoroughly mixed. Due to the strong denaturing conditions cells are quickly lysed,simultaneously RNases are inactivated and the RNA is secured. The RNA is secured. DTT isadded to inactivate the RNases by cleaving intramolecular disulfide bridges.Binding and removal of DNAUnder these conditons DNA during lysis is bound to Lysis Solution S contained carrier.Lysate will be completely transferred to an InviTrap ® 96 Micro Filter Plate and centrifuged. DNAremains bound to the surface of the membrane of the DNA-Binding-Plate A. The RNA-containingsolution is located in the eluate.Binding total RNAAfter adding Binding Buffer R to the eluates, those are completely transferred onto theRNA – Binding Plate and the RNA is bound on the membrane.Removing residual contaminantsContaminants are efficiently washed away using Wash Buffer R1 and R2, while the RNAremains bound to the membrane.ElutionTotal RNA is eluted from the membrane using 60 - 80 µl Elution Buffer R (RNase free water). Theeluted RNA is ready for use in different subsequent tests.7 InviTrap ® RNA Cell HTS 96 Kit/ C and V 0810
Important notesImportant points before starting a protocolAfter receiving the kit, check the kit components for damage. If buffer bottles are damaged, contactthe Invitek Technical Services or your local distributor. In case of liquid spillage, refer to “Safetyinformation” (see page 5). Do not use damaged kit components, since their use may lead to poorkit performance.Sample quantityThe InviTrap ® RNA purification procedure is optimized for the use of 50 to max. 5 x 10 5 cells.Which cell amount shouldn’t exceed, therefore direct cell counting is recommend. Table 1 may beused as a guide.Growth area and number of HeLa cells in various cell-culture platesCell culture plate Growth area in cm 2 * Number of cell**96 well48 well12 well6 well0,3 – 0,61.04.09,54-5 x 10 41,3 x 10 55.0 x 10 51,2 x 10 6*) Growth area varies slightly depending on the supplier**) Confluent growth is assumed. Values are reported per wellThe well membranes have a maximum binding capacity of approx. 100 µg, but actual yields will bedifferent depending on the cell line even for the same amount of cells.If more than 5 x 10 5 cells are to be processed, it is recommend to split the sample. Doubling theused buffer volumes prior RNA binding onto the membrane, doesn’t’ lead to designated result ineither case, particularly with respect to removal of contaminating DNA. Volumes of Wash- andElution Buffer needn’t to be aligned.Depending on used cell line, lysates may become viscous using large cell quantity and this maycause clogged membranes. High Viscosity always leads to lower yield and quality of isolated RNA.Therefore it is recommend to perform preliminary viscosity analysis of the desired cell linedepending on cell quantity, as well as analyzing RNA yield and purity. More than 1 x 10 6 cellsshould not be used.Average total RNA yield depending on cell line using the InviTrap® RNA Cell HTS 96 Kit.Cell Line Source yield in µg for 1 x 10 5 cellsHeLaJurkatMRC 5NIH3T3human cervical carcinomahuman T-cell leukemiahumanhuman2.01,75.06,58 InviTrap ® RNA Cell HTS 96 Kit/ C and V 0810
Page 4: Kit content of InviTrap ® RNA Cell
Page 7: Product characteristic of the InviT
Page 11 and 12: Equipment and reagents to be suppli
Page 13 and 14: Scheme of the InviTrap ® RNA Cell
Page 15 and 16: 16. Add again 400 µl Wash Buffer R
Page 17 and 18: 11. Repeat this washing step by add
Page 19 and 20: 13. Centrifuge at 9.000 x g (6.200
Page 21 and 22: TroubleshootingProblem Cause Commen
Page 23 and 24: When measuring RNA samples, make su
Page 25 and 26: Invitek distributorsAustraliaMedica

RNA Cell HTS 96 Kits

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