Presentation on theme: "Isolation and Purification of Nucleic Acids: Sample Processing"— Presentation transcript:
1Isolation and Purification of Nucleic Acids: Sample Processing Donna C. Sullivan, PhDDivision of Infectious DiseasesUniversity of Mississippi Medical Center
2Outline Principles for handling clinical specimens Types of specimens Fundamentals of specimen handlingNucleic acid preparationDNA isolation methodsRNA isolation methodsMethods of analysis of nucleic acids
3Principles for Handling of All Clinical Specimens Observe universal precautions for biohazards.Use protective gowns, gloves, face and eye shields.Decontaminate all spills and work areas with 10% bleach.Dispose of all waste in appropriate biologic waste containers.Use gloves. Your RNA depends on it!
4Types of Specimens for the Molecular Diagnostics Laboratory Whole bloodBone marrowPBSC (phoresis product)Serum/plasmaBuccal cellsCultured cellsBlood spotsBody fluidsCSFBronchial lavageAmnioticSemenUrineTissue samplesFresh/frozenParaffin-embeddedHair (shaft/root)
5Fundamentals of Specimen Handling: Specimen Labeling Patient name, date of birth, and medical record numberOrdering physicianType of specimenAccession numberDate and time of collectionLaboratory technician identification (initials)Requested test(s)
6Blood and Bone Marrow Isolation of nucleic acids Collection: Genomic DNARNACollection:Collect in an anticoagulant, mix well but gently to avoid disruption of cells
7Anticoagulants EDTA ACD Heparin Lavender-top Vacutainer Preferred specimenACDYellow-top VacutainerHeparinGreen-top VacutainerInhibits several enzymes used in molecular assays
8Specimen Packaging and Shipping: Blood and Bone Marrow DO NOT FREEZE!!!Protect from temperature extremesOvernight delivery preferredPackaging must comply with shipping rules for bloodborne pathogensProtective containerAbsorbent material in packingSealed container in plastic bagLabeled as Biohazard
9The Effect of Tissue Fixatives on the Purification of Nucleic Acid
10Paraffin-embedded Tissue Sections Genetic testing, infectious disease testing, identity testingFormalin-fixed tissue is suitable.Mercury or other heavy metal fixatives are not acceptable.Tissue sections on glass slides can be used for in situ applications and microdissection techniques.
11Specimen Storage Requirements—DNA Blood, Bone Marrow, Other Fluids 22–25 °C Not recommended (<24 hours)2–8 °C Suitable condition for up to 72 hours–20 °C Not recommendedNOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs). Leukocyte pellet can be frozen for up to 1 year.–70 °C Not recommendedNOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs). Leukocyte pellet can be frozen for >1 year.
12Specimen Storage Requirements — RNA Blood, Bone Marrow, Other Fluids 22–25 °C Not recommended within 2 hours2–8 °C Not recommended within 2 hours–20 °C Not recommended 2–4 weeksNOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs).–70 °C Preferred storage conditionNOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs)
13Nucleic Acid Storage Requirements: Storage of DNA Specimens <4 Months1–3 Years<7 Years>7 Years2–25 °C2–8 °C–20 °C–70 °CNot recommendedRecommendedfor long-termstorage in ethanol
16Nucleic Acid Preparation Sample Source? Whole bloodBuffy coatSerum or plasmaBone materialBuccal cellsCultured cellsAmniocytes or amniotic fluidDried blood spotsFresh or frozen tissue (biopsy material)Sputum, urine, CSF, or other body fluidsFixed or paraffin-embedded tissue
17Nucleic Acid Preparation Other Considerations What is the size or volume of each sample?Amount of DNA or RNA requiredEquipment and tube sizes requiredHow many samples are being processed?Capacity of the centrifugeIsolation method speedIs a high-throughput or automated system available?96-well plate methodsWalk-away or semi-automation
18Nucleic Acid Preparation Choosing an Isolation Method Important factors are:Processing speedEase of useYield of DNA or RNAQuality of DNA and RNA prepared (amplification performance)Shelf life/storage conditionsQuality assurance criteriaCost of preparation
19Basic Steps in Isolating DNA from Clinical Specimens Separate WBCs from RBCs, if necessaryLyse WBCs or other nucleated cellsDenature/digest proteinsSeparate contaminants (e.g., proteins, heme)from DNAPrecipitate DNA if necessaryResuspend DNA in final buffer
20DNA Isolation Methods Liquid Phase Organic Extraction Phenol (50):chloroform/isoamyl alcohol (50:49:1)Lysed samples mixed with above; two layers are formed.Proteins remain at interface.DNA is removed with top aqueous layer.DNA is precipitated with alcohol and rehydrated.Disadvantages:Slow, labor-intensive, toxic (phenol, chloroform)Fume hood required, disposal of hazardous materials required
21DNA Isolation Methods: Liquid Phase Nonorganic Salt Precipitation Cell membranes are lysed and proteins are denatured by detergent (such as SDS).RNA is removed with RNase.Proteins are precipitated with salt solution.DNA is precipitated with alcohol and rehydrated.Advantages:Fast and easy methodUses nontoxic materials, no fume hood required, no hazardous materials disposal issuesProduces high-quality DNA
22DNA Isolation Methods Solid Phase Procedures Uses solid support columns, magnetic beads, or chelating agentsSolid support columns: Fibrous or silica matrices bind DNA allowing separation from other contaminants.Magnetic beads: DNA binds to beads; beads are separated from other contaminants with magnet.Chelating resinsAdvantages:Fast and easy, no precipitation required
23DNA Purification Method Comparison Solid PhaseLiquid Phase(Lyse RBCs)Lyse cells(Protein digestion-ProK)Separate proteins from DNAPrecipitate DNA-alcoholRehydrate DNA(Pre-lyse cells)Apply sampleWashElute DNA
24Basic Steps in Isolating RNA from Clinical Specimens Separate WBCs from RBCs, if necessaryLyse WBCs or other nucleated cells in presence of protein denaturants, RNase inhibitorsDenature/digest proteinsSeparate proteins, DNA, and contaminantsfrom RNAPrecipitate RNA if necessaryResuspend RNA in final buffer
25Precautions for Working with RNA in the Clinical Laboratory RNA is not a stable molecule!It is easily degraded by RNase enzymes.Use sterile, disposable plastic ware (tubes, filter tips) marked “For RNA Use Only”.Always wear gloves and work in a hood whenever possible/practical.Treat liquids with DEPC, except Tris-based buffers.
26RNA Isolation Methods Cesium Chloride Gradient Used mainly to get clean RNA for Northern blotsHomogenize cells in guanidinium isothiocyanate and b-mercaptoethanol solution.Add to CsCl gradient and centrifuge for 12–20 hours; RNA will be at the bottom of tube.Re-dissolve in TE/SDS buffer.Precipitate RNA with salt and ethanol, then rehydrate.Advantage: high qualityDisadvantages: extremely time-consuming, hazardous materials disposal issues
27RNA Isolation Methods Guanidinium-based Organic Isolation Phenol/guanidinium solution disrupts cells, solubilizes cell components, but maintains integrity of RNA.Add chloroform, mix, and centrifuge.Proteins/DNA remain at interface.RNA is removed with aqueous top layer.RNA is precipitated with alcohol and rehydrated.Advantage: faster than CsCl methodDisadvantages: fume hood required, hazardous waste disposal issues
28RNA Isolation Methods Nonorganic Salt Precipitation Cell membranes are lysed and proteins are denatured by detergent (such as SDS) in the presence of EDTA or other RNase inhibitors.Proteins/DNA are precipitated with a high concentration salt solution.RNA is precipitated with alcohol and rehydrated.Advantages:Fast and easy, nontoxicProduces high quality RNA
29Resuspending Final Nucleic Acid Samples Have some idea of expected nucleic acid yield.Choose diluent volume according to desired concentration.Calculating Expected DNA YieldExample:1 X 107 cells X 6 pg DNA/cell X 80% yield= 48 mg DNAResuspend DNA in TE buffer or ultra pure DNAse-free water.Resuspend RNA in ultra pure RNase-free water.
30Nucleic Acid AnalysisDNA or RNA is characterized using several different methods for assessing quantity, quality, and molecular size.UV spectrophotometryAgarose gel electrophoresisFluorometryColorimetric blotting
31Quantity from UV Spectrophotometry DNA and RNA absorb maximally at 260 nm.Proteins absorb at 280 nm.Background scatter absorbs at 320 nm.
32Quantity from UV Spectrophotometry [DNA] =(A260 – A320) X dilution factor X 50 µg/mL[RNA] =(A260 – A320) X dilution factor X 40 µg/mLConcentration = µg of DNA or RNA per mL of hydrating solution
33Quantity from UV Spectrophotometry Calculating Yield Multiply the concentration of theDNA or RNA sample by thevolume of hydrating solution added.Example for DNA: 150 µg/mL X 0.1 mL = 15 µgConcentration from UV Spec. (µg DNA per ml of hydrating solution)Volume of hydration solutionDNA yield
34Quality from UV Spectrophotometry A260/A280 = measure of purity(A260 – A320)/(A280 – A320)1.7 – 2.0 = good DNA or RNA<1.7 = too much protein orother contaminant (?)
35Quality from Agarose Gel Electrophoresis Genomic DNA:0.6% to 1% gel, µg/mL ethidium bromide in gel and/or in running bufferElectrophorese at 70–80 volts, 45–90 minutes.Total RNA:1% to 2% gel, µg/ml ethidium bromide in gel and/or in running bufferElectrophorese at 80–100 volts, 20–40 minutes.
36Lambda DNA cut with Hind III DNA Size from Agarose Gel Electrophoresis: Compares unknown DNA to known size standards100 bp ladder1 kb ladderLambda DNA cut with Hind IIILambda DNA48,500 bp (48.5 kb)12,218 bp23,130 bp9,416 bp6,557 bp4,361 bp2,322 bp2,027 bp517 bp1,636 bp3,054 bp6,018 bp100 bp300 bp600 bp1,000 bp1,500 bp1,018 bp2,036 bp
37DNA Quality from Agarose Gel Electrophoresis High molecular weight band (>48.5 kb)Smearing indicates DNA degradation (or too much DNA loaded).
38DNA Quality from Agarose Gel Electrophoresis Lambda DNAmarkerHuman Whole Blood DNALambda DNA cut with Hind III markerWhole blood genomic DNA
39RNA Size and Quality from Agarose Gel Electrophoresis Size: mRNA may be smaller or larger than ribosomal RNA (rRNA).Quality: High-quality RNA has these characteristics:28S rRNA band : 18S rRNA band = 2:1 intensityLittle to no genomic DNA (high MW band)Note: If 18S rRNA is more intense than 28S rRNA, or if both bands are smeared, RNA degradation is probable.
40Cultured Cell RNA 100 50 25 ng DNA 28S 18S Degraded RNADNA28S18S5S rRNA, tRNA, and other small RNA moleculesmRNA = background smearhigh low MWngGenomic DNA markers
41Storage ConditionsStore DNA in TE buffer at 4 °C for weeks or at –20 °C to –80 °C for long term.Store RNA in RNase-free ultra pure water at –70 °C.
42Troubleshooting Nucleic Acid Preparation Methods Problem: No or low nucleic acid yield.Make sure that ample time was allowed for resuspension or rehydration of sample.Repeat isolation from any remaining original sample (adjust procedure for possible low cell number or poorly handled starting material).Concentrate dilute nucleic acid using ethanol precipitation.
43Troubleshooting Nucleic Acid Preparation Methods Problem: Poor nucleic acid qualityIf sample is degraded, repeat isolation from remaining original sample, if possible.If sample is contaminated with proteins or other substances, clean it up by re-isolating (improvement depends on the extraction procedure used).