Summary
Methoden 2 - samenvatting
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Samenvatting van het vak Methoden 2 gegeven door De Keersmaecker Kim | Dupont Patrick | Goris An | Daelemans Dirk | Koole Michel. Door dit document te studeren behaalde ik een 14/20.
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Methoden 2INHOUD
H3 Analyse van RNA ....................................................................................................................................... 6
3.1 RNA voorzorgen en bewaren ........................................................................................................................ 6
3.2 RNA extraheren ............................................................................................................................................. 7
STAP 1: Lyseren ............................................................................................................................................... 7
Stap 2: isoleren ................................................................................................................................................ 8
3.3 RNA kwantificeren ......................................................................................................................................... 9
3.4 RNA concentreren ....................................................................................................................................... 10
3.5 RNA in vitro aanmaken................................................................................................................................ 10
3.6 RNA reverse transcriptie ............................................................................................................................. 11
3.7 RNA analyseren ........................................................................................................................................... 14
3.7.1 Northern blotting ................................................................................................................................. 14
3.7.2 Kwantitatieve PCR toepassingen .......................................................................................................... 15
3.7.3 In-situ hybridizatie................................................................................................................................ 19
3.7.4 In-situ PCR ............................................................................................................................................ 19
Voorbeeld toepassing: Covid-19 diagnostische testen ..................................................................................... 19
H4 Analyse van eiwitten ............................................................................................................................... 23
4.1. Isoleren van eiwitten .................................................................................................................................. 23
4.1.1. Homogenisatiebuffer .......................................................................................................................... 23
4.2. Aanmaken van eiwitten ............................................................................................................................. 23
4.2.1 Chemische synthese in vitro ................................................................................................................. 24
4.2.2 Enzymatische synthese in vitro ............................................................................................................ 24
4.2.3 In vivo expressie systemen ................................................................................................................... 25
4.2.4 Bereiding van antistoffen ..................................................................................................................... 30
4.3. Concentratiebepaling ................................................................................................................................. 33
4.3.1. UV absorptie........................................................................................................................................ 33
4.3.2. Colorimetrische methoden ................................................................................................................. 34
4.3.3 Fluorimetrische methodes ................................................................................................................... 36
4.4. Concentreren van eiwitten ........................................................................................................................ 37
4.4.1. Uitzouten ............................................................................................................................................. 37
4.4.2. Centrifugale filters ............................................................................................................................... 38
4.4.3. Chromatografie ................................................................................................................................... 38
4.5. Kwantificeren en identificeren van specifieke eiwitten ............................................................................. 38
4.5.1. Western blotting ................................................................................................................................. 38
4.5.2. Antilichaam micro-arrays .................................................................................................................... 39
4.5.3. Flowcytometrie ................................................................................................................................... 40
4.5.4. ELISA (enzym linked immunosorbent assay) ....................................................................................... 40
4.5.5. Radioimmunoassay (RIA) .................................................................................................................... 41
1
, 4.5.6. Immunohistochemie ........................................................................................................................... 42
H5 Microscopie ............................................................................................................................................ 43
5.1. Fluorescentiemicrosopie ............................................................................................................................ 43
5.1.1 Inleiding ................................................................................................................................................ 43
5.1.2 Confocale Microscopie (LSCM/SDCM) ................................................................................................. 43
5.1.3 Light Sheet Fluorescentie Microscopie (LSFM) .................................................................................... 45
5.1.4 2-foton Fluorescentie Microscopie ...................................................................................................... 47
5.1.5 Total Internal Reflection Fluorescentie Microscopy (TIRFM) ............................................................... 47
5.1.6 Samengevat .......................................................................................................................................... 48
5.2 Beeldanalyse ............................................................................................................................................... 49
5.2.1 Beeldopname ....................................................................................................................................... 49
5.2.2 Beeldbewerkingen ............................................................................................................................... 51
5.2.3 Samengevat .......................................................................................................................................... 51
5.3 Superresolutie microscopie......................................................................................................................... 52
5.3.1 Inleiding ................................................................................................................................................ 52
5.3.2 Structured Illumination Microscopie (SIM) .......................................................................................... 52
5.3.3 Single Molecule Localization Microscopie (SMLM-PALM/STORM) ...................................................... 54
5.3.4 Stimulated Emission Depletion Microscopie (STED) ............................................................................ 57
5.3.5 Samengevat .......................................................................................................................................... 59
5.4 Elektronenmicroscopie (EM) ....................................................................................................................... 60
5.4.1 Inleiding ................................................................................................................................................ 60
5.4.2 Scanning Elektronen Microscopie (SEM).............................................................................................. 63
5.4.3 Transmissie Elektronen Microscopie (TEM) ......................................................................................... 65
5.4.4 Enkele voorbeelden.............................................................................................................................. 66
5.4.5 Aanvullende EM toepassingen ............................................................................................................. 66
5.5 Scanning Probe Microscopie (SPM) ............................................................................................................ 68
5.5.1 Near-Field Scanning Optical Microscopy (NSOM) ................................................................................ 68
5.5.2 Atomic Force Microscopy (AFM) .......................................................................................................... 69
5.5.4 Samengevat .......................................................................................................................................... 71
H6 Elektrofysiologie ..................................................................................................................................... 72
6.1 Basisbeginselen ........................................................................................................................................... 72
6.2 Equivalente circuits ..................................................................................................................................... 75
6.3 Intracellulaire meettechnieken ................................................................................................................... 78
6.3.1 Micropipet ................................................................................................................................ 78
6.3.2 Patch clamp .......................................................................................................................................... 80
6.3.3 Voltage/current clamp ......................................................................................................................... 83
6.3.4 Beeldvorming ....................................................................................................................................... 84
6.3.5 Geavanceerde patch clamp metingen ................................................................................................. 85
6.3.6 Planaire patch clamp ............................................................................................................................ 85
6.4 Extracellulaire meettechnieken ......................................................................................................... 86
2
, 6.4.1 Ex VIVO ................................................................................................................................................. 86
6.4.2 In VIVO ................................................................................................................................................. 87
6.5 Optische meettechnieken ........................................................................................................................... 89
6.5.1 Calcium indicatoren ............................................................................................................................. 89
6.5.2 Optogenetica ........................................................................................................................................ 92
6.5.3 Spanningsgevoelige indicatoren .......................................................................................................... 94
6.5.4 Intrinsiek Signaal Optimal Imaging (ISOI) ............................................................................................. 95
H7 In-vivo metingen bij de mens ................................................................................................................. 97
7.1 Ultrasone beeldvorming (US) ...................................................................................................................... 97
7.1.1. Karakteristieken van geluid en geluidgolven ...................................................................................... 97
7.1.2. Interacties tussen ultrasone golven en materie .................................................................................. 98
7.1.3. Apperatuur .......................................................................................................................................... 99
7.1.4. Acquisitie – meetproces .................................................................................................................... 101
7.1.5. Doppler-echografie ........................................................................................................................... 101
7.1.6. Afstandsmetingen ............................................................................................................................. 102
7.1.7. Contrastmiddelen.............................................................................................................................. 102
7.1.8. Biologische effecten .......................................................................................................................... 103
7.1.9. Voor- en nadelen van ultrasone beeldvorming ................................................................................ 104
Samengevat: principe van ultrasone beeldvorming ................................................................................... 104
7.2 Magnetische resonantiebeeldvorming (MRI) ........................................................................................... 104
7.2.1. Inleiding ............................................................................................................................................. 104
7.2.2. Resonantie ........................................................................................................................................ 106
7.2.3. T1 en T2 relaxatieparameters ........................................................................................................... 108
7.2.4. Basis acquisitieparameters ................................................................................................................ 109
7.2.5. Spin echo (SE) .................................................................................................................................... 110
7.2.6. Inversion recovery (IR) ...................................................................................................................... 112
7.2.7. Gradiënt echo (GE of GRE) ................................................................................................................ 112
7.2.8. Signaallokalisatie ............................................................................................................................... 113
7.2.9. k-ruimte ............................................................................................................................................. 116
7.2.10. Spatiale resolutie en contrast ......................................................................................................... 117
7.2.11. Angiografie (MRA) ........................................................................................................................... 117
7.2.12. Functionele MRI (fMRI) ................................................................................................................... 119
7.2.13. Diffusie MRI ..................................................................................................................................... 120
7.2.14. Spectroscopie .................................................................................................................................. 122
7.2.15. Veiligheid ......................................................................................................................................... 123
7.2.16. Voor- en nadelen MRI ..................................................................................................................... 123
Samengevat: principe van MRI beeldvorming ............................................................................................ 123
7.3 Interactie tussen straling en materie ........................................................................................................ 124
7.3.1. Elektromagnetische straling .............................................................................................................. 124
7.3.2. Rayleigh verstrooiing ......................................................................................................................... 124
3
, 7.3.3. Compton verstrooiing ....................................................................................................................... 124
7.3.4. Foto-elektrisch effect ........................................................................................................................ 125
7.3.5. Paar productie ................................................................................................................................... 127
7.3.6. Dominante interactie ........................................................................................................................ 127
7.3.7. Lineaire ATTENUATIE coëfficiënt....................................................................................................... 128
7.4 X-stralen beeldvorming ............................................................................................................................. 128
7.4.1. introductie ......................................................................................................................................... 128
7.4.2. X-stralen productie ............................................................................................................................ 129
7.4.3. X-stralen detectie .............................................................................................................................. 130
7.4.4. Fysisch CT principe en beeldreconstructie ........................................................................................ 131
7.4.5. CT acquisities ..................................................................................................................................... 132
7.4.6. CT angiografie ................................................................................................................................... 133
7.4.7. CT perfusie ........................................................................................................................................ 133
7.4.8. Cardio CT ........................................................................................................................................... 133
7.4.9. Dual source CT................................................................................................................................... 133
7.4.10. Photon-counting CT ........................................................................................................................ 134
7.4.11. Voor- en nadelen CT ........................................................................................................................ 134
Samengevat: principe van CT beeldvorming ............................................................................................... 134
7.5 Productie van radio-isotopen .................................................................................................................... 135
7.5.1. Inleiding ............................................................................................................................................. 135
7.5.2. Radioactief verval .............................................................................................................................. 135
7.5.3. Half-leven t1/2 van een radionuclide .................................................................................................. 138
7.5.4. Productie van radionucliden ............................................................................................................. 139
7.6 Single foton emissie tomografie (SPECT) .................................................................................................. 140
7.6.1. Inleiding ............................................................................................................................................. 140
7.6.2. Gamma camera ................................................................................................................................. 140
7.6.3. Computer tomografie (reconstructie) ............................................................................................... 142
7.6.4. Partieel volume effect ....................................................................................................................... 142
7.6.5. Attenuatiecorrectie ........................................................................................................................... 142
7.6.6. Verstrooiingscorrectie ....................................................................................................................... 143
7.6.7. Toepassingen..................................................................................................................................... 143
7.6.8. Nieuwe ontwikkelingen ..................................................................................................................... 144
7.6.9. Voor- en nadelen SPECT .................................................................................................................... 144
7.7 Positron emissie tomografie (PET) ............................................................................................................ 145
7.7.1. Inleiding ............................................................................................................................................. 145
7.7.2. Coïncidentie detectie ........................................................................................................................ 146
7.7.3. Detectoren ........................................................................................................................................ 146
7.7.4. Correcties .......................................................................................................................................... 147
7.7.5. Computer tomografie (reconstructie) ............................................................................................... 148
7.7.6. Kinetische modellering ...................................................................................................................... 148
4
, 7.7.7. Nieuwe ontwikkelingen ..................................................................................................................... 150
7.7.8. Voor- en nadelen PET ........................................................................................................................ 150
Samenvatting PET ....................................................................................................................................... 150
7.8 Principes van radiotherapie ...................................................................................................................... 151
7.8.1. Interactie van deeltjes met materie .................................................................................................. 151
7.8.2. Stralingsbiologie ................................................................................................................................ 153
7.8.3. Radiotherapie .................................................................................................................................... 155
7.8.4. Protontherapie .................................................................................................................................. 156
7.9 Radioprotectie ........................................................................................................................................... 157
7.9.1. Inleiding ............................................................................................................................................. 157
7.9.2. deterministische en stochastische effecten ...................................................................................... 157
7.9.3. Effectieve dosis ................................................................................................................................. 158
7.9.4. Bescherming ...................................................................................................................................... 159
7.9.5. Risico classificatie .............................................................................................................................. 159
H8 Radio-isotooptechnieken in het labo .................................................................................................... 161
8.1. Beeldvorming bij kleine proefdieren ........................................................................................................ 161
8.2. Radioimmunoassay (RIA) ......................................................................................................................... 161
5
,H3 ANALYSE VAN RNA
DNA versus RNA
3 belangrijke verschillen → hebben tot gevolg dat er verschillen zijn in detectiemethoden die we gebruiken
(nucleobasen, dubbelstreng versus enkelstreng, deoxyribose versus ribose)
Vraag: waar hebben we dideoxyribose gezien? → ddNTPs bij Sanger sequencing om ketenverlenging te stoppen,
3’OH vormt phosphodiesterverbindingen
2’OH van RNA: is meer vrij om te reageren, bv door spontane hydrolyse of door enzymen (RNAse)
DNA: meestal dubbelstrengs in dubbele helix, RNA kan ook dubbele helix vormen maar meestal enkelstrengs,
wel secundaire en tertiaire structuur
Evolutionair gezien is DNA ontstaan uit RNA, uracil-houdend DNA bestaat in hele zeldzame organismen (fagen)
maar thymine is beter aangepast om DNA onveranderd te houden (beter bewaard)
Daarnaast is RNA korter
Soorten RNA
3 grote categorieën RNA die te maken hebben met DNA-RNA-eiwit paradigma:
(1) mRNA (1-4%)
(2) rRNA (niet coderend), ribosomaal RNA, verschillende subunits (80-50%)
(3) tRNA (niet coderend), transfer RNA, fysische link tussen mRNA en eiwit
Steeds meerdere klassen van niet-coderend RNA ontdekt (overzicht groeiend, long coding en short coding RNA,
enkel belang voor Methoden)
Korte non-coding RNAs
- miRNA: eerst ontdekt in worm C elegans, daarna ontdekt dat die ook bij de mens voorkomt, in kaart gebracht
dankzij kennis over humane genoom sequentie vanaf 2003, nu >2000 miRNAs gekend, rol in gezondheid en in
ziekte
- piRNA: piwi-interacting RNA, interageert met Piwi eiwitten, vooral belangrijk in kiemcellen en dus in
vruchtbaarheid, minder bestudeerd
- siRNA: small interfering RNA, short interfering RNA or silencing RNA → ook gebruikt als methode voor het
bestuderen van genexpressie
Long-noncoding RNA:
- lncRNA: >16,000 lncRNAs gekend
- circular RNA: nog maar heel recent ontdekt (2012-2017)
Belang voor Methoden → korter, geen poly-A staart, in veel minder kopieën voorkomend in cellen (minder
abundant) → aangepaste methoden nodig om te extraheren of identificeren voor groeiend onderzoek naar deze
types RNA
Structuur mRNA
Uiteinden van mRNA: niet gecodeerd in DNA maar toegevoegd
5’cap: gemodificeerde G (methylgroep en dubbele fosfaatbrug)
Poly-A tail: 50-250 A’s aan het uiteinde
pre-mRNA: bestaat niet lang, niet makkelijk te meten, vlak na transcriptie (bevat nog wel intron)
3.1 RNA VOORZORGEN EN BEWAREN
Mogelijke problemen
- RNA profiel verandert na staalname, vb inductie genen
- RNA degradatie door endogene (in staal) of exogene (van buitenaf) RNAsen, vb RNAse A
- DNA contaminatie: scheiding DNA/RNA is meestal niet 100% mogelijk
Staalname
Voorkeur EDTA als anticoagulans (heparine inhibeert DNA/RNA reacties)
RNA stabilisatie om degradatie en inductie te voorkomen
6
, - Onmiddellijke verwerking
- Invriezen vloeibare stikstof -196°C
- Totaal bloed in vacuüm tube met RNA bewaarmiddel, vb PAXgene tube
- Cellen/weefsel met een RNA bewaarmiddel, vb RNAlater (percipiteert eiwitten) of Trizol (eerste stap
van extractie)
Staalverwerking (RNAse-vrij werken)
- Zeer zuiver werken
- Labojas en handschoenen om stalen te beschermen (tegen RNAse op huid)
- Afgebakende labo/bench-zone voor RNA werk
- Oppervlakken, pipetten, …: RNAse schoonmaakproduct, vb RNAseZAP (alle RNAse verwijderen)
- RNAse vrije filter-pipettips en plastics
- Nuclease-vrije oplossingen/water:
• Gefilterd water (Milli-Q), RT-PCR grade water (getest op RNAsen)
• Behandeling met RNAse inhibitoren door DEPC (diethylpyrocarbonaat, carcinogeen):
reageert met amines in aminozuren, vooral histidine, in de katalytische site van RNAsen en
inhibeert deze (Tris = amine → DEPC werkt niet in Tris-buffer)
• Nieuwere RNAse inhibitoren (minder carcinogeen)
RNA bewaring
- RNAse-vrij water (of TE-buffer 10/1, maar EDTA inhibiteert RNAse niet, en DNAsen wel)
- Bewaring tot 1 jaar op -80°C (minder lang dan DNA, minder stabiel)
- Langere bewaring eventueel als alcoholprecipitaat
- Beter: meerdere aliquots staal in RNA bewaarmiddel voor latere RNA extractie bewaren
DNA contaminatie
Moeilijk volledig DNA vrij te krijgen maar is niet altijd een probleem, kijken naar wanneer toepassing onderscheid
maakt tussen DNA/RNA
Detecteren
- RT min controle
- Elektroforese (zie RNA kwantificeren)
Verwijderen: DNAse behandeling
- Toevoegen tijdens RNA extractie of behandeling van al geëxtraheerd RNA
- DNAseI (knipt dsDNA en ssDNA) of dsDNA-specifieke DNAsen
- Inactivatie (hitte) of verwijdering DNAse na behandeling
- Nadeel: verlies of degradatie van RNA mogelijk, daarom niet altijd gebruikt (niet standaard)
3.2 RNA EXTRAHEREN
Methode is afhankelijk van → Startmateriaal (weefsel, bloed, bacteriecultuur, virussen,...)
→ Doel (hoeveel nodig, welke kwaliteit,…)
3 essentiële stappen
(1) Lyseren Weefsel/cellen afbreken zodat RNA vrijkomt, lysaat is vloeistof met inhoud van gelyseerde
cellen, goede homogenisatie belangrijk
(2) Isoleren RNA uit het lysaat halen of opzuiveren (zonder eiwitten, eventueel zonder DNA)
(3) Oplossen RNA in gewenste type en hoeveelheid vloeistof oplossen, daarbij concentreren
STAP 1: LYSEREN
7
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