I loaded my RNA samples, ran the gel for 5 hours and this was the result:
Arrraagh!!!!!!
It appears that I have RNase contamination in my gel! This is an enzyme found in abundance on skin (and in saliva and tears) that specifically destroy RNA, hence the horrible smeary bands you see. RNase can be found on almost every surface in physical contact and is hell to get rid of. For a better description - and for how I and many others who work with RNA feel about this enzyme - check out this page or just take me at my word - Evil! Evil! Evil!
RNA stands for ribonucleic acid and is one of three types of macromolecule (the other two being DNA and protein) that are essential for all forms of life. RNA is very similar to DNA in that it is made up of a string of nucleotides, the sequence of which allows RNA to encode genetic information, similar and as derived from DNA. However, unlike DNA, different types of RNA molecule exist, and are involved in many enzymatic processes within the living cell: that as well as encoding and passing on the information to synthesise proteins, RNA can direct and regulate gene expression, function in protein synthesis and sense and respond to cellular signals.
An x-ray structure of transfer RNA - a type of RNA that is involved in recruiting and linking amino acids together in the process of protein synethesis. Image by Yikrazuul.
Yet despite being so deeply involved in many vital biological processes RNA is very unstable. This is not surprising since a lot of these processes are rapidly occurring and are responsive to internal and external factors. RNA molecules generated now may not appropriate for what may occur at a later stage; their prolonged presence may even be deterimental to the cell in that specific catalytic processes may be overexpressed or suppressed; certain functions may be deregulated. One can say instead of being cogs in a well oiled machine they are now like spanners in the works.
So, yes, it is good that RNA is easily degradable and RNase is in abundance to do it - just not when one is trying to isolate and study it!!! So it will be take-two tomorrow: I am going to encase my hands in chain-mail gloves and treat all my equipment and my bench with a flame thrower. Hasta la vista RNase...
Kidding!!!
Wish me luck!
Hi there! This is a good read. I will be looking forward to visit your page again and for your other posts as well. Thank you for sharing your thoughts about rnase in your area. I am glad to stop by your site and know more about rnase. Keep it up!
ReplyDeleteRNase A is an RNase that is commonly used in research. RNase A (e.g., bovine pancreatic ribonuclease A: PDB 2AAS) is one of the hardiest enzymes in common laboratory usage; one method of isolating it is to boil a crude cellular extract until all enzymes other than RNase A are denatured. It is specific for single-stranded RNAs. It cleaves the 3'-end of unpaired C and U residues, ultimately forming a 3'-phosphorylated product via a 2',3'-cyclic monophosphate intermediate.
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.