The EXTRACTME TOTAL RNA kit is designed for the rapid and efficient purification of high quality RNA from 1-30 mg of tissue (fresh or frozen) and 104-107 cultured cells. The isolation protocol and buffer formulations were optimized for high isolation efficiency and purity of RNA.
- fresh or frozen tissue: 1-30 mg
- tissue preserved in RNase inactivating buffers: 1-30 mg
- cell culture: 104-107 cells
The typical efficiencies of RNA isolation from fresh biological material are given in section XIII.
approx. 90 μg RNA
- 16-20 minutes (lysis and homogenisation time not included)
- 30-60 minutes for homogenization in liquid nitrogen
- 30-40 minutes for mechanical homogenization (ceramic beads)
A260/A280 ratio = 1.9 – 2.1
The EXTRACTME TOTAL RNA kit utilises spin minicolumns with membranes which efficiently and selectively bind nucleic acids at high concentration of chaotropic salts. The isolation procedure consists of 6 steps. In the first isolation step, the tissue is homogenized in order to disintegrate intercellular bonds (epithelial tissue) and fragmentize high-molecular proteins (muscle or connective tissue). Then the homogenate is lysed with guanidine thiocyanate and detergents. Any RNases are inactivated by guanidine thiocyanate and β-mercaptoethanol. The homogenate is separated from the undigested tissue/cell remains by centrifugation and on the Homogenizing Column. The RNA is bound to the Purification Column membrane by addition of ethanol. The three-step washing stage effectively removes impurities and enzyme inhibitors. The purified RNA is eluted using a low ionic strength buffer or RNase-free water (pH 7.0-9.0) and can be used directly in all downstream applications such as RT-PCR, Northern blotting, RT-qPCR and so forth.
The quality of each production batch (LOT) of the EXTRACTME TOTAL RNA kit is tested using standard QC procedures. The purified RNA concentration, quality and stability are evaluated by gel electrophoresis and spectrophotometer. In addition, the functional quality is tested by reverse transcription and qPCR.
Manufacturer: BLIRT S.A.