MagZol Reagent is a reagent system for the isolation of total RNA from cells and tissues. The reagent, a single-phase solution consisting of phenol and guanidine isothiocyanate, is modification of the single-step RNA isolation method developed by Chomczynski and Sacchi. The sample is homogenized and lysed in MagZol Reagent which maintains the integrity of the RNA, while disrupting and denaturing endogenous RNases and other cellular components. Extraction of the lysate with chloroform further denatures proteins and separates the mixture into an organic and an aqueous phase. RNA remains exclusively in the aqueous phase, and is subsequently recovered by isopropanol.
Detail
Introduction
MagZol Reagent is a reagent system for the isolation of total RNA from cells and tissues. The reagent, a single-phase solution consisting of phenol and guanidine isothiocyanate, is modification of the single-step RNA isolation method developed by Chomczynski and Sacchi. The sample is homogenized and lysed in MagZol Reagent which maintains the integrity of the RNA, while disrupting and denaturing endogenous RNases and other cellular components. Extraction of the lysate with chloroform further denatures proteins and separates the mixture into an organic and an aqueous phase. RNA remains exclusively in the aqueous phase, and is subsequently recovered by isopropanol.
This method is suitable for small quantities of tissue (<100mg) and cells (<5 X106), and large quantities of tissue (up to 1g) and cells (<108), of human, animal, plant, or bacterial origin. The simplicity of the MagZol Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total RNA prepared in this manner can be used for Northern blot analysis, dot blot hybridization, poly(A) + selection, in vitro translation, RNase protection assay, and molecular cloning. For use in amplification by thermal cycling, treatment of the isolated RNA with RNase-free DNase I is recommended when the two amplimers lie within a single exon.
Details
Specifications
Features
Specifications
Main Functions
Extract RNA from liquid samples by salting out method
Applications
RT-PCR, Northern hybridization, poly (a) enrichment, etc.
Purification technology
Acid phenol guanidine isothiocyanate
Process method
Manual (centrifugation)
Sample type
Various liquid samples
Sample amount
Flexible
Elution volume
Variation with sample size
Time per run
Variation with sample size
Advantages
Flexible – sample amount can be adjusted according to the demand
Cost performance -the most economical nucleic acid extraction technology
Storage and Stability
MagZol Reagent should be stored at 2-8°C upon arrival and is stable for at least 24 months under the condition. However, short-term storage (up to 12 weeks) at room temperature (15-25°C) does not affect its performance.
Other Products
APOPercentage apoptosis assay kit
Product Info
Document
Product Info
What is Apoptosis?
Apoptosis is an essentially normal physiological process that removes now redundant, cells, particularly during embryonic development and early growth. In adult animals the process removes cells that are irreparable. The apoptotic process is also involved in many major diseases such as cancer, where transformed tumour cells have their apoptotic process disabled, permitting cell cycling to continue unchecked. In contrast some forms of senile dementia may result from excessive apoptotic induction of neural cells.
The apoptotic process in mammalian cells is a rapid event (2‐4 hours). Within this short time span an apparently viable cell can be quietly dismantled, to disappear leaving no visible trace of its former existence.
How is apoptosis detected or measured?
An apoptosis cascade of activators, effectors and regulators has been identified. This in turn led to a range of apoptosis assays being devised to detect and monitor these events. Some laboratories will employ two distinct assays, one selected to detect early (initiation) apoptotic events, while a second assay will target a later (execution) event. Apoptosis assays, based on methodology, can be classified into four major inter‐linked groups:
[1] DNA fragmentation (electrophoresis and nick end labelling, TUNEL).
[2] Apoptotic proteases (fluorescently labelled antibodies to the caspases).
[3] Flow cytometric analysis (FACS, incorporating other group assays).
Biocolor’s APOPercentage assay is based on the latter. Further information can be found under the ‘Mode of Action’ Tab.
How does APOPercentage detect apoptosis?
The mammalian cell membrane has been described as a semi‐fluid mosaic structure, composed of phospholipids with a diverse group of inserted proteins and some cholesterol. The phospholipids are the major components of the membrane and are arranged in the form of a ‘bi‐layer’; which is asymmetric in composition, structure, and function.
To ensure normal transmembrane functions the phospholipids must be maintained in an asymmetric composition. The process is regulated by ‘flippases’, which catalyse the active transport of aminophospholipids from the outer to inner monolayer. However, in cells undergoing apoptosis, flippase is overwhelmed by the action of another enzyme, termed ‘floppase’ or ‘scramblase’. The net effect is a scrambling of the phospholipid distribution between the inner and outer monolayers.
Cell membrane changes during apoptosis
The APOPercentage assay utilises an intense, pink-coloured dye reagent which is taken up during in-vitro culture by apoptosis-committed cells. This uptake occurs at the stage of Phosphatidylserine transmembrane movement, as produced by the flipflop mechanism. Dye uptake continues until blebbing occurs. No further dye can then enter the now defunct cell and the dye that has accumulated within the cell is not released (unlike necrotic cells which release dye).
Since the dye reagent is excluded or not retained by healthy or necrotic cells it therefore acts as a specific label for apoptotic cells.
How are APOPercentage-labelled cells quantified?
Labelled apoptosis cells may then by conveniently analysed by the following methods:
Direct Analysis The intense pink colour of the labelled cells can be visually assessed using brightfield microscopy. Apoptosis in substrate-adherent cell populations is therefore readily quantified using image analysis techniques. This technique is the most sensitive with the ability of detecting one single apoptotic cell per well.
Colorimetry protocol Dye that accumulates within apoptotic cells is released into solution via addition of Dye Release Reagent. The concentration of this intracellular dye is then measured at 550nm using a microplate colorimeter/spectrophotometer.
NB: The APOPercentage assay kit does NOT require the use of a Flow Cytometer.
Limit of Detection
A single cell (via image analysis method)
Detection Method
Colorimetric (550nm) (Endpoint) or Image Analysis based
Measurements per kit
Sufficient for 4×24 well plates or 6×96 well plates
Suitable Samples
Adherent mammalian cells (in-vitro)
APOPercentage kit contents:
1. APOPercentage Dye (1x5ml)
2. Dye Release Reagent (1x150ml)
3. Phosphate Buffered Saline (PBS) (1x120ml)
4. 24-well starter plate.
5. Assay kit manual.
The Colorimetric Protocol requires a Microplate Colorimeter / Spectrophotometer.
Additional 96-well plates will be required for use when reading dye absorbance values.
The Direct Detection Protocol Requires an inverted stage microscope with an attached digital camera.
NB: Additional reagents (typically culture medium and suitable apoptosis treatments) may be required for sample preparation prior to assay. Consult manual or contact us for further details.
Document
The APOPercentage™ Apoptosis kit is a dye-based, colorimetric assay for detection and measurement of apoptosis (programmed cell death) during in-vitro cell culture.
Convenient optimized on-column DNase treatment using Norgen’s RNA Purification Kits
Also includes protocol for digestion in-solution followed by RNA Clean-Up
Guaranteed RNase-Free
Includes Enzyme Incubation Buffer
Cat. 25710 contains one vial of 1,600 units and Cat. 25720 contains 4 vials (1,600 units/vial)
Norgen’s RNA purification kits isolate total RNA with minimal amounts of genomic DNA contamination. However, for some sensitive downstream applications, it may be desirable to remove all traces of residual DNA. Norgen’s RNase-free DNAse I Kit, with Enzyme Incubation Buffer, can be used for optional on-column DNase digestion with any of Norgen’s RNA purification kits. Alternatively, after isolating total RNA using one of Norgen’s RNA purification kits, the RNA elution can be treated with this DNase I. The RNA can then be purified from the DNase using Norgen’s RNA Clean-Up and Concentration Kit (Cat# 23600), and the RNA can then be used in downstream applications.
Details
Each RNase-Free DNase I Kit is supplied complete with sufficient enzyme and enzyme incubation buffer for 50 or 200 reactions.
Storage Conditions The DNase I provided is in lyophilized form. It is stable for at least 3 months if stored at room temperature. However, it is recommended to store the DNase I vial at 2 – 8ºC (or below) upon receipt to maintain stability beyond 3 months. Buffer DR and Enzyme Incubation Buffer can be stored at room temperature. After reconstitution with Buffer DR (see product manual), the DNase I should be stored at -20ºC. All reagents should remain stable for at least 1 year in their unopened containers at the appropriate storage temperature.
