The density of 70% ethanol allows RNA settlement or say sedimentation in the vial.
I have not personally used the Qiagen Total RNA Extraction Kit for RNA extraction.
"b -mercaptoethanol is used to help to destroy RNases that may be present and will degrade the RNA. b -mercaptoethanol is a reducing agent that will reduce the disulfide bonds of the RNases, thereby destroying the conformation and the functionality of the enzyme". It comes from http://www.norgenbiotek.com/index.php?id=faqs_rnakits
QIAzol Lysis Reagent is used to lyse cells and tissues to release RNA for extraction. It disrupts the cellular and nuclear membranes, thus allowing the RNA to be isolated and purified from the lysate.
Adjusting the pH to 7 during RNA extraction helps to create the optimal conditions for RNA stability. RNA is more stable at a neutral pH, which minimizes degradation and helps maintain the integrity of the RNA molecules during the extraction process. This ensures that high-quality RNA is obtained for downstream applications.
in RNA extraction we don't need to use a strong lysis solution to the cells like in DNA extraction since we don't need to break the nuclear envelope in case of RNA*. *Be cautious, in some case (ex. hnRNA) the RNA is in the nucleus so you have to break it. Really depend on what you are looking for.
75% ethanol is commonly used in RNA extraction because it helps to wash the RNA pellet by removing salts and other contaminants, while also helping to maintain the integrity and stability of RNA molecules. The lower ethanol concentration reduces the risk of RNA degradation and allows for efficient RNA recovery during the extraction process.
Ethanol is commonly used in DNA extraction because it effectively precipitates DNA from aqueous solutions, allowing for easy separation and purification. In RNA extraction, isopropyl alcohol is preferred because it provides higher yields and better purity of RNA, which is more sensitive and prone to degradation. Additionally, isopropyl alcohol helps to minimize the co-precipitation of contaminants and proteins, ensuring a cleaner RNA sample.
I have not personally used the Qiagen Total RNA Extraction Kit for RNA extraction.
This wash step allows you to centrifuge the sample and collect a "clean" RNA pellet, after discarding the supernatant that contained contaminating salts and proteins. When isolating and purifying RNA, 75% ethanol is used as a wash solution because RNA is a precipitate (solid) in this percentage of ethanol, while most proteins and salts remain in solution (are soluble). At a lower % ethanol, both the RNA and the proteins would be soluble, so you would not be able to separate them. At a higher % ethanol, both the RNA and salts would remain in the pellet, so you would not be able to separate the salts from your RNA. Prior to the wash step, you probably added 100% ethanol to your sample, so the final total concentration of ethanol was 75%. This step is where the RNA precipitates out of solution. You would then centrifuge the sample and discard the supernatant, as above. In the wash step, you are merely using the same solution (75% ethanol) to wash the RNA pellet you created in the previous step.
"b -mercaptoethanol is used to help to destroy RNases that may be present and will degrade the RNA. b -mercaptoethanol is a reducing agent that will reduce the disulfide bonds of the RNases, thereby destroying the conformation and the functionality of the enzyme". It comes from http://www.norgenbiotek.com/index.php?id=faqs_rnakits
Ethanol is commonly used in microbiology labs as a disinfectant to sterilize surfaces, equipment, and lab benches. It is also used for flame sterilization of inoculating loops and needles. Additionally, ethanol is used in DNA and RNA extraction protocols to precipitate nucleic acids.
Chloroform is commonly used in RNA extraction to separate RNA from other cellular components. It helps in the denaturation of proteins and the dissolution of lipids during the extraction process. Chloroform aids in the formation of a distinct organic phase where RNA can be collected.
QIAzol Lysis Reagent is used to lyse cells and tissues to release RNA for extraction. It disrupts the cellular and nuclear membranes, thus allowing the RNA to be isolated and purified from the lysate.
Ethanol is often used in laboratory settings to precipitate DNA and RNA during the extraction process, though its role in mitosis is not direct. In the context of cell division, ethanol can induce cell cycle arrest, particularly by affecting the spindle apparatus and disrupting normal mitotic progression. This disruption can lead to apoptosis or changes in cell viability, making ethanol useful in research for studying cell division and its regulation. However, high concentrations of ethanol can be toxic to cells, affecting their ability to properly undergo mitosis.
RNAse destroys the RNA and hence RNAse contamination is a problem in RNA extraction as it breaks down RNA. RNAse enzyme is removed by using RNAse inhibitor or precautions like wearing of gloves, autoclaving tips , using RNAse free water/DEPC treated water is done while performing RTPCR
Adjusting the pH to 7 during RNA extraction helps to create the optimal conditions for RNA stability. RNA is more stable at a neutral pH, which minimizes degradation and helps maintain the integrity of the RNA molecules during the extraction process. This ensures that high-quality RNA is obtained for downstream applications.
Proteinase K is used to digest proteins in a sample, making DNA or RNA more accessible for extraction. Buffer AL is used to help inactivate Proteinase K after digestion, to ensure it does not interfere with downstream applications. Together, they are commonly used in molecular biology techniques like DNA or RNA extraction from various samples.