Working with RNA

Ribonucleic Acid Ribonucleic acid (RNA) is a polymer consisting of nucleotides containing ribose and four different nucleotides adenine, guanine, cytosine, and uracil. Three main classes of RNA molecules are transcribed from DNA by three different types of RNA polymerases mRNA, tRNA and rRNA. Other RNA types are found in very small amounts including snRNAs, double-stranded RNA (dsRNA) and other non-coding RNAs such as the SRP RNAs. All RNA classes present in cells serve different functions. 

Messenger RNA (mRNA) is synthesized by the transcription of DNA and transported to the cytoplasm where it serves as a template for the synthesis of proteins. Ribosomal RNA (rRNA) is a part of the ribosomes, which consist of rRNA and proteins. Eukaryotic ribosomes contain four different rRNA molecules 18S, 5.8S, 28S, and 5S rRNA. rRNA makes up to 80% of the RNA molecules. 

Transfer RNA (tRNA) molecules are small molecules composed of 74—95 nucleotides (nt). The function oftRNA is to deliver amino acids required for protein synthesis to the ribosomes. RNAs are also a part of riboproteins, involved in RNA processing. 

Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA and found in Eukaryotic cells. The dsRNA forms also the genetic material of some viruses. 

A typical mammalian cell contains 10-30 pg of total RNA. The majority of RNA molecules are tRNAs and rRNAs, which have a relatively long half-life. mRNA accounts for only 1-5% of the total cellular RNA. Some mRNA species have a half-life of minutes whereas others can have a half-life of many days. Some mRNAs comprise as much as 3% of the mRNA pool whereas others account less than 0.01%. Approximately 350000 00000 mRNA molecules are present in each single mammalian cell, made up of approximately 12000 different transcripts with an average length of approximately 2 kb (Tables 3.7, 3.8). 

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Skin, too, has add-ons that increase its effectiveness as a barrier. In a biochemistry laboratory you often have to wear gloves to protect yourself from the material you re handling, but sometimes you have to wear gloves to protect the material from you. People who work with RNA wear gloves because human skin excretes an enzyme that chops up RNA. Why It turns out that many viruses are made from RNA. To such a virus, the enzyme is like razor wire on the skin any RNA that tries to breach the barrier gets lacerated. 

CRITICAL ASSESSMENT OF THE METHOD Since differential expression analysis means to compare the quantities of RNA species in two samples, every step during sample preparation has to be highly reproducible. In order to maximize reproducibility, complete total RNA extraction in a one step procedure is recommended. Care must always be taken when working with RNA, to avoid contamination with RNA ases, which may result in RNA degradation. 

This book is divided into five chapters. Chapter 1 is an introduction to the characteristics of RNA-protein interactions based on models that have emerged from the plethora of technologies available today. Chapter 2 describes the specific precautions recommended for working with RNA and outlines the most commonly... 

Sterile, disposable polypropylene tubes are recommended for working with RNA. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases. In ordertodestroy RNases, non-disposableplastic-warecan be rinsed withO.l M NaOH, 1 mM EDTA, followed by RNase-free water. Alternatively, chloroform-resistant plastic-ware can be rinsed with chloroform, which is sufficient to inactivate RNases. 

Whichever method of purification is chosen, other precautions to eliminate adventitious RNase contamination should also be employed, especially by the less practiced worker. Even the most experienced worker occasionally suffers from the degradation of RNA preparations associated with RNase contamination. This can be from many sources and even in some cases appear to be an act of God. When problems are encountered, or if you are not accustomed to working with RNA, the following precautions should be employed. 

Compared with DNA, more care has to be taken when working with RNA to prevent any RNase contamination. Use of gloves (hands are the major source of RNase), diethyl pyrocarbonate (DEPC)-treated solutions, disposable tubes, pipet tips from freshly opened bags, and RNasin (a potent ribonuclease inhibitor) in the labeling reaction, minimizes the risk of RNA degradation. 

Citrate works with metals that work with RNA Jack Szostak s lab, working with protocells made from simple membranes, found that magnesium in the protocells would actually start to cut apart RNA strands. This problem went away when citrate was added to the mix, because the citrate helped moderate the bad magnesium chemistry. Remember that citrate is central to the citric acid cycle and works well with iron, so it would have been very useful for early biochemical cycles. Here again, adding more early-Earth chemicals makes for more successful reactions. 

The proteins that work with RNA show apattern of evolution... L. Li et al. Aminoacylating urzymes chsdlenge the RNA world hypothesis. 2013. / Biol Chem. 288(37), p. 26856. DOI 10.1074/jbc.M113.496125. 

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