Product Description

RNA Extraction Reagent has been composed to isolate and purify RNA of highest quality, yield, purity, and repeatability for downstream applications. Using reagent RNA can be isolated from biological samples of different sources e.g. human, other animals, plants, yeast, bacterial and viral origin, etc. reagent is a monophasic reagent having phenol, guanidine isothiocyanate and other proprietary components.

Product Specifications

Reagent is highly efficient in isolating large and small molecular sized RNA. It can maintain the integrity of RNA during sample homogenization as it inhibits RNase activity during lysis of cells and other cell organelles. After homogenization of samples in presence of RNAZOL RNA  Extraction Reagent, chloroform is added to the solution. After incubation and centrifugation, aqueous phase is separated carefully. The bright Yellow colour of the reagent makes the phase separation (Yellow colored organic phase and colorless aqueous phase) clearly visible. RNA is precipitated using isopropanol, followed by washing in 75% ethanol. The samples must be air dried before eluting in RNase free DEPC water.

Reagent is highly efficient in isolating large and small molecular sized RNA. It can maintain the integrity of RNA during sample homogenization as it inhibits RNase activity during lysis of cells and other cell organelles. After    homogenization of samples in presence of RNAZOL RNA  Extraction Reagent, chloroform is added to the solution. After  incubation  and centrifugation, aqueous phase is separated carefully. The bright Yellow colour of the reagent makes the phase separation (Yellow colored organic phase and colorless aqueous phase) clearly visible. RNA is precipitated using isopropanol, followed by washing in 75% ethanol. The samples must be air dried before eluting in RNase free DEPC water.

Compatible Downstream Applications

• Molecular Cloning
• RT-qPCR, RT-PCR
• cDNA Synthesis
• Northern Blot analysis
• poly(A)+ Selection
• Other allied applications

Usage Protocol

RNA isolation protocol using reagent has the following steps: Sample Preparation – Lysis

Lysis of samples must be done in presence of  Reagent

• Tissue: 1mL of Reagent to be added to 50-100mg of animal tissue sample and homogenized. For plant sample (ex: leaf), crushing of plant material to powder in liquid nitrogen is to be carried out prior to adding Reagent.

Note – Fresh sample must be cleaned with RNAse blaster (Cat No: 411103 Field samples must be stored in RNAGuard to ensure RNA integrity

• Monolayer Cells: Firstly,  growth media to be removed. Secondly, 0.5mL of RNAZOL is added to 1 x 10 ⁵– 10⁷ cells.
• Cell Suspension: Cells (can be of bacteria, animal, plant or yeast origin) are firstly pelleted down and supernatant discarded. 1mL of RNAZOL is added to the pellet followed by lysis and homogenization.

Phase Separation

• The samples must be incubated in RNAZOL Reagent for 5 minutes at room temperature for complete dissociation of nucleoproteins
• 200 µL of chloroform to be added for every 1 mL of RNAZOL and incubated for 2-3 minutes
• 200 µL of chloroform to be added for every 1 mL of RNAZOL and incubated for 2-3 minutes
• A distinct three-layer phase is formed in the solution mix, an upper aqueous phase, interphase and lower Yellow organic phase
• The upper aqueous phase is collected in a fresh microcentrifuge tube. The upper aqueous phase is ≥50% of the total volume

Precipitation & Washing of RNA

• 700 µL of Isopropanol is added to the upper aqueous phase, mixed well and incubated at room temperature for 5 minutes.
• The solution is then centrifuged at 12000 rpm for 15 minutes at 4⁰C. A jelly like precipitate will be observed on the side and bottom of the tube
• Supernatant is carefully removed. The precipitate is washed using 1 mL of 75% Ethanol at 7500 rpm for 5mins at 4⁰C. Supernatant is carefully discarded.

RNA Elution

• The pellet obtained after washing is air dried at room temperature to remove all the remaining ethanol
• The pellet is then resuspended in 20-50 µL of RNase free water (DEPC Water) and the sample is incubated at 55⁰C for 5-10 minutes
• Purity of isolated RNA is checked using Spectrophotometer and Agarose gel (≥ 1.5%). RNA is ready for downstream application or must be stored at -80°C for future use.

Additional Materials and Equipment:

The below listed materials and equipment’s should be arranged by the user prior to initiating RNA extraction:

• Autoclaved & RNase free Microcentrifuge tubes
• Pipettes
• Autoclaved & RNase free pipette tips
• Vortex
• Centrifuge
• Water/Heat Bath
• Chloroform
• 100% isopropanol (Mol. bio grade)
• 75% Ethanol (Prepared with DEPC Water)
• DEPC Water