PRINCIPLE

The EXTRACTME TOTAL RNA KIT utilizes spin minicolumns with membranes which efficiently and selectively bind nucleic acids at high concentration of chaotropic salts. During the first isolation step, a tissue is homogenized in order to disintegrate intercellular bonds (epithelial tissue) and fragmentize high-molecular proteins (muscle or connective tissue). A homogenate is lysed with guanidine thiocyanate and detergents. RNases are inactivated by guanidine thiocyanate and ß-mercaptoethanol (optional). The homogenate is separated from undigested tissue/cell that remains after centrifugation. RNA bounds to a Purification Column membrane by addition  of ethanol. On-column DNase digestion enables removal of remaining genomic DNA. A three-step washing stage effectively removes impurities and enzyme inhibitors. Purified RNA is eluted with the use of low ionic strength buffer or RNase-free water and may be used directly in all downstream applications, such as RT-PCR, RT-qPCR, Northern blotting, cDNA synthesis, primer extension, RNA sequencing, microarrays.

PRODUCT SPECIFICATIONS

SAMPLE MATERIAL

• fresh or frozen tissue : up to 30 mg
• tissue preserved in RNase inactivating buffers (e.g. RNAlater®, Ambion): up to 30 mg
• cell culture: up to 10⁷ cells

BINDING CAPACITY

~ 230 μg RNA

TIME REQUIRED

• 10–12 minutes (lysis and homogenization time not included)
•  15–30 minutes for homogenization in liquid nitrogen
• 15–20 minutes for mechanical homogenization (ceramic beads)
• 5 minutes for optional DNase I treatment

RNA PURITY

A₂₆₀/A₂₈₀ ratio = 1.9 – 2.1