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.
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.
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.
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
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.
Cold ethanol is most likely used instead of room temperature ethanol in order to prevent the ethanol from evaporating. When the temperature of something decreases the molecules speed decreases as well making it less likely to evaporate.
In a DNA extraction, the purpose of a buffer is to solubilize DNA as well as RNA. Because of this, it prevents the DNA for degrading.