E.Z.N.A.^® Blood RNA Midiprep Kits are designed for the isolation of total cellar RNA from up to 10 ml of fresh whole blood or whole blood treated with common anticoagulants such as heparin, EDTA, or acid-citrate-dextrose. 10 ml of blood typically yields 10-50µg of total RNA. The procedure completely removes contaminants and enzyme inhibitors making total RNA isolation fast, convenient, and reliable. The kit is also suitable for isolation of total RNA from body fluids, tissues, and from RNA viruses.

Principle

The E.Z.N.A^® Blood RNA Midiprep Kits use the reversible binding properties of HiBind^® matrix, a new silica-based material., combined with the speed of mini column spin technology. Red blood cells are selectively lysed and white cells collected by centrifugation. After lysis of white blood cells under denaturing conditions that inactivate RNases, total RNA is purified on the HiBind^® spin column. A specially formulated high salt buffer system allows RNA molecules greater than 200 bases to bind to the matrix. Cellular debris and other contaminants (such as hemoglobin) are effectively washed away and high quality RNA is finally eluted in DEPC treated sterile water. RNA purified using the E.Z.N.A.^® Blood RNA method is ready for applications such as RT-PCR.

Procedure

Red blood cells are lysed by mixing ERL buffer to fresh whole blood. Centrifugations pellets white blood cells facilitating the removal of lysed red blood cells. White blood cells are re-suspended in ERL buffer and TRK lysis buffer is added to the white blood cells. The re-suspended cells are homogenized to efficiently extract RNA. An equal volumes of ethanol is added to the to the lysate to precipitate RNA and facilitate binding to the spin column. Samples are applied to HiBind^® RNA Midi spin-column and subjected to multiple wash steps to remove any impurities present in the RNA. Total RNA is then eluted from the column with DEPC-treated water.

Note : To completely remove genomic DNA, use the on column DNase treatment.

Applications

RNA purified using the E.Z.N.A^® Blood RNA Midiprep Kit can be used in a number of applications including :

• RT-PCR