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

RECOMMENDATIONS AND IMPORTANT NOTES

Sampling and storing the material for RNA isolation

Proper sampling and storing of biological material, prior to RNA isolation is crucial to obtain a high purity RNA. After sampling, the material should be preserved by deep freezing (at -80°C or in liquid nitrogen) or stored at -20°C in RNase inactivating buffers (e.g. RNAlater®, Ambion). Most tissues should obligatory be preserved within 30 minutes of sampling. Tissues rich in RNases (pancreas, liver) require an immediate preservation.

While isolating from cell cultures, best results are achieved with the use of fresh material. If storage is unavoidable, discard the supernatant after centrifugation and freeze the cell pellet at -80°C or in liquid nitrogen.

RNase elimination

RNases are very active enzymes which do not require any cofactors and are resistant to 15 minutes autoclaving at 121°C. In order to avoid enzyme’s degrading effect on RNA, it is essential to follow the recommendations below:

1. Use disposable gloves at all times when working with RNA. Do not come in contact with any items that are not specifically designed to work with RNA.
2. If possible, keep the samples at 2–8°C at all stages of the procedure, including centrifugation. Use decontaminated freezing racks instead of ice in order to avoid RNase contamination. Keeping RNA, after elution, in the freezing racks is recommended.
3. Plastic disposables (tips, tubes) should be RNase-free or autoclaved at 134°C for 18–20 minutes.
4. Reusable plastic, glass and porcelain should be soaked overnight in 0.1 N NaOH/0.1% DEPC water (or RNase-free water) and then washed with 0.1% DEPC water (or RNase-free water). When applicable, glass and porcelain (mortars) should be parched at 150–140°C for 2–4 h and cooled to room temperature.
5. Wipe surfaces, pipettes, centrifuge (rotor should be wiped separately) and tube racks with 3% hydrogen peroxide or <0.5% sodium hypochlorite (or any commercially available RNase inactivating fluid). Prior to  decontamination, test the decontaminant on a small area of the material for possible undesired reactions.

DNA contamination

All biological material used for RNA isolation contains DNA. There is no RNA isolation method that may guarantee a complete DNA removal unless RNA sample is treated with DNase after isolation. Even a slight DNA contamination (several gDNA copies per reaction) may give an additional signal in a quantitative PCR analysis after reverse transcription. The EXTRACTME TOTAL RNA KIT allows efficient on-column digestion of DNA during RNA purification as a optional step. DNase I can be removed by RW1 Buffer.