The Total RNA Kit I provides a rapid and easy method for the extraction of up to 100 µg of total RNA from cultured eukaryotic cells and tissues. The kit allows for the simultaneous processing of samples in less than 25 minutes. Normally up to 1 x 107 eukaryotic cells, 1 x 10⁹ bacterial cells, or 40 mg tissue can be used in a single experiment. While this kit may be used for isolation of RNA from whole blood, we recommend use of the Blood RNA Kit (Catalog #R6614-01/02) as it is specifically designed for effective hemolysis and hemoglobin removal and gives higher RNA yields. If processing samples that contain higher levels of fat in their composition, we recommend using the Total RNA Extraction Kit II (Catalog # R6934-01/02) as it is formulated to deal with high fat content.

Principle

The Total RNA Kits use the reversible binding properties of HiBind^® matrix, a new silica-based material., combined with the speed of mini column spin technology. A specially formulated high salt buffer system allows more than 100 µg of RNA molecules greater than 200 bases to bind to the matrix. Cells or tissues are first lysed under conditions that inactivate RNases. Lysed samples are subsequently applied to the HiBind^® spin columns to effectively bind total RNA, enabling wash steps which remove virtually all possible contaminants. High quality RNA is finally eluted in DEPC-treated sterile water. 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

Cells or tissues are combined with TRK Lysis Buffer and homogenized 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 a wash step with RNA Wash Buffer I. At this point an optional on column DNase I digestion reaction can be applied to if high purity RNA is required (Catalog # E1091-01/02). These processes are followed by additional washes with RNA Wash Buffer II. After completion of the wash steps RNA is eluted with DEPC-treated water.

Applications

RNA purified using the Total RNA method can be used in applications such as :

• RT-PCR
• Northern Blotting
• Nuclease Protection
• In vitro translation
• Poly A+ RNA (mRNA) purification

Denaturing formaldehyde agarose gel electrophoresis of total RNA.
RNA was isolated by Total RNA Kit from mouse Liver (1),
Hela Cells (2), and mouse tissue (3).

1-2 X 10e5 LNCaP cells were extracted with either the E.Z.N.A. total RNA kit or 1 ml of Trizol and prepared
according to manufacturers instructions. The RNAs were quantitated on the Nanodrop spectrophotometer
and showed that the Trizol gave better total yields. Subsequently, 1 ug of each treatment RNA was treated
with amplification grade DNaseI (from Invitrogen), reversed transcribed using the Invitrogen Superscript III
kit and finally treated with RNaseH to remove residual RNA. cDNA was then subjected to 31 cycles of PCR
with primer pairs that produced a 534 bp amplicon for beta-actin (lane 2 and 6), a 350 bp amplicon for
GAPDH (lanes 3 and 7) and a 350 bp amplicon for beta-actin (lanes 4 and 8). Lanes 2-4 are the E.Z.N.A. cDNA
results while lanes 6-8 are the Trizol cDNA results. You can clearly see the smearing that occurred from
contaminants in the cDNA prepared from Trizol derived RNA while no such contamination is present in the
cDNA prepared from the E.Z.N.A.-derived RNA. Also, the primers for the 350 bp GAPDH and beta-actin
amplicons have clearly yielded more product in the E.Z.N.A. lanes.