RNA Extraction Basic Practical Knowledge. Kis Enikő OKK-OSSKI PCR training course June 13-17, 2016
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1 RNA Extraction Basic Practical Knowledge Kis Enikő OKK-OSSKI PCR training course June 13-17, 2016
2 RNA Only one strand more vulnerable than two-stranded DNA. Ribose-phosphate backbone Uracil in place of thymine Different RNA pool in the nucleolus, the nucleus or in the cytoplasm. Complicated secondary and terciary structures for free energy minimalization.
3 Nature of information one can obtain through cell RNA content investigation: Gene expression-regulation information Alternative spliced transcripts investigation Gene fusion Epigenetic regulation Transpozone regulation
4 Types of RNA in the Cell Micro-RNA - mirna: identification code denotes mrnas which are to be decoyed for the ribonuclease protein complex (22-24b) Small interferring RNA sirna: post-transcriptional silencing and methylation of DNA target sites; heterochromatin formation (22-24b) PIWI-interacting RNA pirna: in the cytoplasm they are RNA decomposers, while in the nucleus they draw DNA or histone methylation; they have a role in transposon mrna breakdown (22-24b) Transfer RNA trna: amino acid transport to the ribosomes during protein biosynthesis (76-90b) Small nucleolar RNA snorna: rrna maturation (around 100b) Small nuclear ribonucleic acid snrna: mrna maturation (around 150b) Long non-coding RNA - lncrna: transcription regulation (around200b) Messenger RNA mrna: template for protein biosynthesis ( b) Ribosomal RNA rrna: translation/protein synthesis (t.l. 7216b)
5 mrna : facts that must be taken into consideration: Procaryotes: polycistronic more proteins from the same mrna: at the same time more ribosomal complexes work on the same, just maturated mrna which on the other end might simultaneously be decayed by a protein complex Eucharyotes: the majority is monocistronic mrna one protein one mrna mrna presence in cells gene expression analysis Transcription is time-dependent and abundance of mrna in a cell depends on protein necessity and stimulus. Very variable between different cells in nature and quantity from fewer than 10 copies to several hundreds of copies good quality RNA isolate is needed for further investigations.
6 Choosing a well working method
7 Major Steps of RNA Isolation Using Membrane-based Assays Rnase removal Homogenization of sample/cell lysis Lysate filtration RNA precipitation Bind RNA to a membrane DNA digestion Cleaning RNA Dry membrane RNA elution Quality and quantity controle RNA storage
8 The Dreadful RNases Endoribonucleases or exoribonucleases. They are everywhere. RNase7 is secreted by human skin as antipathogene defense. Some tissues and cells contain them in abundant measures (eg. pancreas, some fruits) Reactivate after boiling or autoclaving.
9 How to protect one s RNA from them?
10 Protection Against RNases We usually never isolate DNA and RNA in the same areas/labs. Equipment used for RNA extraction is cleaned thoroughly and kept separate from common lab equipment. We treat tables, racks and platforms with various harsh chemicals (eg. DEP, formamide, chloroform, NaOH and SDS combined solution) that destroy RNases. Always use and often change gloves. Even for preparation of labware and chemicals. At the beginning of the extraction RNA is exposed to RNases released from the sample. Use of different Rnase inhibitor reagents like β-mercapto-etanol, guanidine isothiocyanate, phenol or SDS in our solutions insures intrinsic protection of RNA. At the end of the procedure special care is required because in pure water RNA is very vulnerable. RNA is immediately placed on ice and frozen to -20 C or /especially in case of important samples/ -70 C.
11 Determining the Correct Amount of Starting Material Depends: RNA content of sample Lysis buffer capacity The capacity of the method (eg: phenol-chloroform method vs. membrane based method) kits usually emphasize this information Usually around 1*10 7 cells or 10-30mg tissue sample would work.
12 Homogenization Methods Total disruption of the cell walls and plasma membranes of cells are required Different samples different methods Homogenisation shears high molecular weight DNA and cellular components, reduces wiscosity of the sample Manual: douncer (Alexander Dounce ) glass mortar with ceramic pestle - 13µm cells are wrecked but not organelles. (eg. plants, filametous funghi) Mechanical: scalpel or syringe-and needle (eg. animal cell monolayers) Automatic homogenizator (eg. animal tissues) Bead homogenizer (plants, cells or tissues) Enzimatic (eg. yeast) Cryopulverization Incomplete homogenisation: Lower amounts of RNA Incomplete binding to the membrane
13 Homogenization of Sample and Cell Lysis - Considerations Tissue sections Cultured cells: Easier to lyse no/little homogenization required Needle and syringe Homogeneous RNA pool Different lysis difficulties due to ECM composition Homogenizers: manual, mechanincal or automated Directly homogenise frozen samples Smaller sample requisite Heterogeneous RNA pool Hard to reproduce
14 Cell Lysis Strong denaturating agents: Total RNA extraction Releases all nucleic acid content Proteins are also denaturated and inactivated. Rnases, too. Dnase treatment required Tissue lysis also feasible. Mild lysis buffer: Keeping cell compartements intact Separated isolation of different RNA pools of different compartments Attention to RNases! Intrinsic inactivators are inevitable to use.
15 Purifying RNA Sample filtration might be needed in case of insufficient homogenization. RNA precipitation is usually performed by etanol or isopropanol. Binding RNA to a membrane facilitates purification/ otherwise it is imperial not to loose the precipitate. RNA binds strongly to the silica membrane in presence of high salt concentration, and strong denaturing agents (eg.: guanidium salts). DNA digestion by DNase I very sensible to physical denaturation and functionates at room temperature. After centrifugation and washing steps, the nucleic acid can be washed off with low salt aqueous solution!
16 RNA Concentration and Quality Control Quantity might be determined by: spectrophotometric quantification: 260nm: 1U corresponds to 40µg/ml Multi-Mode Microplate Reader: at 260/280nm using the Gene5. program / Nucleic Acid Quantification RNA quality control: 260/280nm: <1.8 protein contamination. Optimal: 2.00 ( ) 260/230nm: <1.8 other organic contaminants. Optimal: /240nm: <1.4 ionic contamination. Optimal: 1.4 Gel electrophoresis: agarose gel at a 1.2% concentration. For mammalian rrna, a 28S:18S rrna ratio of 2:1 is generally representative of goodquality RNA. Special RNase free and denaturating formaldehyde agarose gel electrophoresis. Bioanalyser: software-based electrophoresis analysis
17 RNA Quality Control 2. Phenol-chloroform method Membrane-based on-column isolation
18 Storage Short term (<1 week) dissolved in nuclease-free water or alcoholic precipitate at -20 C. Long-term storage (2-6 months): dissolved in nucleasefree water at -80 C. In pure formamide (100%), FORMAzol or RNAlater up to two years at -20 C, BUT: it must be cleaned prior to reverse transcription! Aliquoting samples is very useful in case of multiple downstream use to avoid thaw-freez cycles
19 Thank You for Your attention!
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