Introduction
E.Z.N.A® Blood RNA Kits are designed for isolation of total intracellular RNA from up to 1 ml of fresh, whole blood treated with any common anticoagulant such as heparin, EDTA, or acid-citrate-dextrose. One milliliter of blood typically yields 1–5 µg 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 cultured cells, tissues, and from RNA viruses in serum or plasma.
Principle
The E.Z.N.A® Blood RNA 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.
The use of mini-columns facilitate the binding, washing, and elution steps thus enabling multiple samples to be simultaneously processed. For more information on columns, click Flexible Multi-Format Columns.
Procedure
Red blood cells are lysed by mixing ERL buffer to fresh whole blood. Centrifuge to pellet the Leukocytes and discard the supernatant containing the lysed red blood cells. Wash the white blood cell pellet again with ERL buffer and add TRK lysis buffer to the pelleted white blood cells. Homogenize to efficiently extract RNA. Equal volumes of ethanol are added to the to the lysate to precipitate RNA and facilitate binding to the spin column. Samples are applied to HiBind® RNA spin column and subjected to multiple wash step (once with Wash Buffer I and twice with Wash Buffer II). Centrifuge the empty column for 1 minutes at full speed to dry the column matrix and elute the RNA with DEPC-treated water.
Note : To remove genomic DNA completely, treat the eluted RNA with RNase-free DNase.
Applications
RNA purified using the E.Z.N.A® Blood RNA Kit can be used in a number of applications including :
