RNA itself difficult to handle because of its unstable nature. Unlike DNA, RNA has free 2'-OH group in their ribose sugar that make them highly reactive. Other than this, RNAse contamination is everywhere (during isolation RNAase from our skin can kill RNA).
I have not personally used the Qiagen Total RNA Extraction Kit for RNA extraction.
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.
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.
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.
Seventy percent ethanol is commonly used in RNA extraction to wash and remove salts and contaminants from the RNA sample. It helps to purify the RNA by precipitating it out of the solution while leaving behind impurities. Additionally, the 70% ethanol concentration helps minimize RNA degradation during the extraction process.
Molecules that can interfere with DNA extraction include proteins, polysaccharides, lipids, and polyphenols. These molecules can bind to DNA, causing it to be more difficult to extract or making the DNA susceptible to degradation during the extraction process. It is important to use appropriate methods to remove or inhibit these molecules before extracting DNA from cells.
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 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.
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
"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
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.
If the DNA is not pure, contaminants include RNA and proteins