RNA Extraction Methods PDF

What is RNA ? Ribonucleic acid (RNA) is a molecule that is present in the majority of living organisms and viruses. It is made up of nucleotides, which are ribose sugars attached to nitrogenous bases and phosphate groups. Types of RNA  POST TRANSCRIPTIONAL MODIFICATIONS Eukaryotic RNA...

What is RNA ? Ribonucleic acid (RNA) is a molecule that is present in the majority of living organisms and viruses. It is made up of nucleotides, which are ribose sugars attached to nitrogenous bases and phosphate groups. Types of RNA  POST TRANSCRIPTIONAL MODIFICATIONS Eukaryotic RNA Processing 5′ Capping 3′ Poly-A Tail Pre-mRNA Splicing  When an RNA transcript is first made in a eukaryotic cell, it is considered a pre-mRNA and must be processed into a messenger RNA (mRNA). A 5' cap is added to the beginning of the RNA transcript, and a 3' poly-A tail is added to the end. In splicing, some sections of the RNA transcript (introns) are removed, and the remaining sections (exons) are stuck back together.  RNA splicing The third big RNA processing event that happens in your cells is RNA splicing. In RNA splicing, specific parts of the pre-mRNA, called introns are recognized and removed by a protein-and-RNA complex called the spliceosome. What are the "good parts"? The pieces of the RNA that are not chopped out are called exons. The exons are pasted together by the spliceosome to make the final, mature mRNA that is shipped out of the nucleus.  What is the Need for Post-Transcriptional Modification? Capping: The capping of pre-mRNA is carried out by the addition of 7- methylguanosine to the 5'end. This is achieved by the removal of terminal 5' phosphate that is done with the help of phosphate enzymes. This reaction is accelerated by the enzyme adenosyltransferase, which at last produces diphosphate 5’end. The process of capping on the primary transcripts gives stability to the molecule, also prevent the mRNA from degradation by the ribonucleases present in the cytoplasm. Last but not least, the caps also help in the recognition of the mRNA by the translation machinery. Tailing: The polyadenylated tail gives extended stability to the pre-mRNA. It further signals the end of transcription to the cellular factors for transportation of the transcript to the cytoplasm. Splicing: The process of splicing helps in evolution by creating new combinations of exons. It also helps in the regulation of gene expression and protein content of the cell.  Transcription in prokaryotes Transcription is a continuous process and occurs in the cytoplasm. Only one type of RNA polymerase is used to synthesize RNA molecules. No post-transcriptional modifications occur, and the mRNA is devoid of a 5' guanosine cap and a poly A tail.  Transcription in prokaryotes Transcription in eukaryotes Transcription is a continuous process Transcription is a separate process and occurs in the cytoplasm. and occurs in the nucleus. Three types of RNA polymerase Only one type of RNA polymerase is enzymes are involved in the eukaryotic used to synthesize RNA molecules. transcription process. No post-transcriptional modifications The mRNA produced has RNA cap at occur, and the mRNA is devoid of a 5' the 5' end and a poly-A tail at the 3' guanosine cap and a poly A tail. end. Importance of RNA Isolation Sequence Size 01 03 measure cloning 02 expression 04 levels Methods of total RNA Isolation (1) Organic Extraction = Steady-state RNA Isolation (2) Affinity Purification = Nuclear run-off Isolation (1) Steady-state RNA Isolation Cell Lysis o Lysis buffer De-Proteinization o Trizol Precipitation o Isopropanol Washing o Ethanol 70% Resuspension o RNase free or DEPC treated water  Components used in RNA Extraction Trizol: is a monophasic solution of phenol & chloroform + guanidinium isothiocyanate. ✓ Acidic phenol/ chloroform Partitioning of RNA into aqueous supernatant for separation ✓ Guanidinium isothiocyanate Powerful protein denaturant Inactivation of RNases. Aqueous phase: RNA Interphase: DNA Organic phase: Lipids, Proteins  Components used in RNA Extraction Diethylpyrocarbonate (DEPC): Irreversibly reacts with the catalytic amino acid in the reactive centre of the RNAse molecule, inhibiting the enzyme. DEPC is a suspected carcinogen and should be handled carefully  ▪ Compatible with a variety of sample types ▪ Can process small and large samples. ▪ Inexpensive. ▪ Organic solvents ▪ Not high-throughput. ▪ RNA may contain contaminating genomic DNA. (2) Affinity Purification  ADVANTAGES o Eliminates need for organic solvents o Compatible with a variety of sample types (tissue, tissue culture cells, white blood cells, plant cells, bacteria, yeast, etc.) o DNase treatment eliminates contaminating genomic DNA. o Excellent RNA purity and integrity.  Messenger RNA Isolation Combine cytoplasmic RNAs Poly-A tail of mRNA binds rRNA and tRNA is and oligo(dT) matrix under to oligo(dT) matrix washed away hybridization conditions Purified mRNA is eluted from the oligo(dT) matrix in water or low-salt buffer  RNA (UNSTABLE!!!) ▪ RNA is an unstable molecule and has a very short half- life once extracted from the cell or tissues. ▪ RNA is especially unstable due to: 1. RNA single stranded. 2. RNA contains ribose (with hydroxyl group). These hydroxyl groups make RNA less stable than DNA because it is more prone to hydrolysis. 3. ubiquitous presence of RNases.  RNases ▪ Ribonuclease (RNase): is a type of nuclease that catalyzes the degradation of RNA into smaller components. o Present in blood, all tissues as well as most bacteria and fungi in the environment. o Can be difficult to inactivate.  Successful RNA isolation depends on 1. Suppression of endogenous RNases. 2. Avoid contamination with exogenous RNases during extraction. (1) Suppression of Endogenous RNases ▪ Samples should be processed immediately or stored at -70 OR -80 degree until required. ▪ Inactivation of RNases by strong denaturing agents like urea, guanidinium hydrochloride, guanidinium isothiocyanate. (2) Avoid contamination with Exogenous RNases during extraction ▪ Specify glassware, solutions, equipment to be used for RNA extraction only. ▪ Treat water and laboratory utensils with diethylpyrocarbonate (DEPC) which is a strong RNAase inhibitor. ▪ Autoclave glassware, solutions and equipment if possible. ▪ Use disposable gloves, disposable plastic materials that must be RNAase free.  Estimating RNA quality Spectrophotometer Gel electrophoresis  Electrophoresis ▪ DNA and RNA carry negative charges. ▪ After extraction, they are subjected to horizontal denatured electrophoresis in agarose gel. ▪ Ethidium bromide or SYBR green dyes are added to the gel. ▪ Ethidium bromide inserts itself between the nucleotides of DNA or RNA and fluoresce under ultraviolet light.  Quantification ▪ Quantification of extracted nucleic acids is done by using spectrophotometer as follows: ▪ RNA sample absorbance are determined on the spectrophotometer at 260nm, 280nm. o 260nm: nucleic acid (DNA, RNA, nucleotides) o 280nm: protein ▪ So, the concentration of extracted RNA in a sample OD at 260 x 40 x dilution factor= [RNA] μg/ml.  Estimating RNA purity by spectrophotometry ▪ A260/A280 ▪ Pure RNA will exhibit an A260/A280 ratio within the range of 1.8 - 2.0. ▪ If the RNA exhibits a ratio lower than 1.7, this indicates ▪ Protein contamination in your sample ▪ If the RNA exhibits a ratio higher than 2, this indicates ▪ Organic solvent contamination in your sample Question: RNA sample (undiluted) is measured and the A260 reading is OD=1.2, the A280 reading is OD=0.7. Calculate the following: ✓ A. RNA concentration ✓B. RNA purity ✓C. RNA yield THANK YOU