This preview shows page 1. Sign up to view the full content.
Unformatted text preview: Western blots Where the heck do I find out what these blots should look like? Review articles on collagen Papers that have done similar Western blots Product information sheets for the antibodies 1 Collagen I Made up of two different subunits 1(I) and 2(I) 1(I) is the larger of the two; both are ~95kD (but can show up higher than that on the blot) Monomers 1(I) and 2(I), dimers [1(I)]2 and 1(I)2(I), and trimer [1(I)]22(I). 2 Procollagen bands The procollagen chains form a trimer The N and C propeptides are cleaved off, forming mature collagen Multiple mature collagens form 3 Lecture 8 RNA Purification, Quantification, and Analysis; RT-PCR "Genetics explain why you look like your father, and if you don't, why you should." 4 Challenge: Record/draw/diagram in as much detail as possible the protein synthesis pathway everything between DNA and protein.
5 Gene-Protein Expression 6 RNA Extraction, Quantification, and Purity Week 8 Part A 7 Proteins vs. RNA
Lysis for Protein Protein Collection (lysis) Total Protein Quantification (BCA) Specific Protein Detection (Western blot) Specific Protein Quantification (Image J) Lysis for RNA mRNA Collection (lysis/purification) Total mRNA Quantification (260nm) Specific mRNA Detection (RT-PCR + agarose gel) Specific mRNA Quantification (requires special techniques like Real Time PCR or a DNA microarray)
8 Western blotting vs. RT-PCR
Protein Analysis: What has been made? Half-life of proteins in the body varies wildly Enzymes in the cell hours mRNA Analysis: What is being made? mRNA has a short half-life (20-60 minutes) Ornithine decarboxylase: 0.2 hours cytochrome c: 150 hrs Extracellular matrix in the body - months to years
9 Protein vs. mRNA Analysis
How are these questions important to tissue engineering? A functional tissue needs to have the proper components to mimic necessary chemical and material properties Ideally, a tissue needs to maintain these properties (by continued protein production or other adaptation) An engineered tissue that's perfect today may not be perfect in a week or a month unless the cells inside it are maintaining it properly
10 Analysis of mRNA All methods of mRNA analysis require very good technique in the first step: RNA extraction and isolation Avoid contamination with Ribonucleases (RNases) - which are everywhere 11 RNases Proteins designed to hydrolyze mRNAs Seriously, they're everywhere! Sweat Hair Skin Dust 12 Protecting your RNA Samples Glassware Plastic ware Gloves Reagent bottles and tubes Solutions Temperature Designated area
13 Glass and metal 14 RNA exposed to RNase Bake at 180 oC overnight Autoclaving is not good enough Plastic Use sterile plastic ware and machinepackaged pipette tips. Pour tubes from an unopened bag (or bag marked For RNA Use Only) onto an RN'ase-free surface Surfaces can be made RN'ase-free by wiping down with a 0.5 M NaOH solution. A sheet of clean tinfoil or plastic wrap will also work
Solutions should be treated with 0.1% DEPC (diethyl pyrocarbonate) DEPC is suspected to be a carcinogen and should be handled with only in a fume hood. Add 0.1% DEPC and shake vigorously Autoclave for 15 minutes at 15 lb/sq. in. on liquid cycle or incubate at least 12 hours at 37 C and then heat to 100 C for 15 minutes. DEPC is not appropriate for amine-containing buffers like Tris
16 Temperature Keep RNA samples on ice. Store your purified RNA samples at -20C (short term) or 70 C (long term) 17 RNA Types Ribosomal RNA (rRNA) Regulatory RNAs ~60% RNA/40% protein 5S: ~150 nucleotides 23S: ~2,900 nucleotides Mostly <25 nucleotides long Heterogeneous size (characteristic) ~3% is mRNA Transfer RNA (tRNA) 18 Week 8a Lab To extract RNA from cultured cells To measure RNA concentration and purity of your extracts To prepare an RNA sample for RT-PCR 19 Week 8a RNA Isolation Wash cells with PBS and lyse with RLT buffer (for RNA lysis) Scrape and homogenize as necessary Add 70% ethanol Purify RNA from other materials in the lysate using mini-spin column 20 Week 8a RNA Isolation 21 Centrifugation Samples must be spun at max speed (>14,000 RPM) The low-speed centrifuges are not adequate When centrifuging for 15 or 30 seconds, add 15 seconds to give the centrifuge time to get to 14,000 RPM
22 Week 8a RNA Isolation Elute the RNA into Rnase-free water Quantify the RNA & determine its purity Dilute sample in a 100 L cuvette Read concentration and 260/280 ratio in the biophotometer
23 RNeasy-Purified RNA Elution contains all RNA molecules longer than 200 nucleotides Most RNAs <200 nucleotides (small rRNAs, tRNA, microRNA, etc.) are selectively excluded RNeasy yield should be ~10-5 ug of RNA/cell RT-PCR with our kit requires 5 g 1 ng RNA per reaction
24 Quantification and Purity Pure RNA absorbs at 260 twice as much as it absorbs at 280 nm Ratio of 1.8 to 2.0 is good Ratio of 1.5 to 1.8 can be acceptable (though not 25 RT-PCR Week 8 Part B 26 Bio-rad PCR Song 27 Now that we have RNA, what do we do with it? Agarose electrophoresis Northern blot RT-PCR Real Time RT-PCR DNA Microarray 28 Agarose electrophoresis Run RNA through formaldehydeagarose gel Stain with Ethidium Bromide (EtBr) or an equivalent DNAbinding dye Streaks indicate RNA degradation 29 Northern blot Southern is for DNA Northern for RNA Western for proteins For each you separate molecules by size with electrophoresis then transfer to solid support (membrane) followed by detection of specific target 30 Northern blotting Run a formaldehyde-containing agarose electrophoresis gel with your RNA samples Capillary transfer of RNA from the formaldehyde gel to a membrane Hybridization of a labeled probe to a specific RNA (the target) 31 Northern Blot
EthBr Anx V GAPDH Stain membrane with labeled oligonucleotides designed to complement the RNA They act just like antibodies in a Western blot
32 Northern blot problems Sensitivity is relatively low Signal is highly sensitive to RNA degradation 33 RT-PCR Reverse Transcriptase Polymerase Chain Reaction All cellular mRNA is reverse transcribed into cDNA PCR is performed on the cDNA to amplify a portion of a single cDNA This exponential amplification boosts the signal! Detects the expression of a specific gene
34 PCR vs. RT-PCR
PCR amplifies DNA (either specific sections of DNA or, using random primers, any DNA present) Determine homology between species Determine homology between genes Detection of mutations Clinical diagnoses Forensic science RT-PCR turns mRNA into cDNA, amplifies part of a single cDNA of interest using PCR Detects whether a gene is on or off
35 RT First strand synthesis mRNA (Collagen II) mRNA (GAPDh) mRNA (EGF receptor) mRNA (TGFalpha) mRNA -> cDNA RNA = template Oligo dT primer RT
cDNA (Collagen II) cDNA (GAPDh) cDNA (EGF receptor) cDNA (TGFalpha) Not selective all mRNA present yield a cDNA Enzyme: Reverse transcriptase 36 First strand synthesis 37 PCR Polymerase chain reaction Method to amplify DNA in a test tube DNA between the sense and antisense primers is exponentially amplified If the primers are random, all DNA will be amplified
3' sense 5' antisense cycle 5' 3' 38 PCR Steps Denature 94 C; double strands break up into single strands Primers attach, and if done right, nothing else does Taq polymerase does its job, beginning at attached primers Anneal Extend Thermocycler Repeat for 25-? cycles
RT-PCR Animation PCR Animation 40 RT-PCR 41 PCR 42 Polymerases Polymerases are proteins Most denature at high temperatures Need to add fresh enzyme after denature step From the bacterium Thermus aquaticus Endures the cycling at high temperatures Taq polymerase is the exception Fun fact: Taq polymerase can polymerize 1000 base pairs in < 10 seconds!
43 Primers Must be specific and efficient in annealing to the target sequence There are programs (like Primer3 or OligoPerfect) that help Finding primers in the literature is a good bet, too
44 Design of Primers Unique sequence (check with BLAST) G-C clamp at 3' end (1-2 G/C nucleotides) Not self-complementary (< 3 contiguous bases) sense ~or~ antisense ~or~ "hairpin turn" Not complementary to the other primer (< 3 contiguous bases)
sense antisense 45 Design of Primers Random base distribution and composition
45-55% G/C content Primer length of ~18-25 bases Distance of intraprimer sequence 100-600 bases apart Equal to product size
5' 3' 3' sense 5' antisense 46 Design of Primers Match primer melting temperature (TM) The primers must work at the same time under the same conditions, so they should anneal at approximately the same temperature Tm (in C) = 2AT + 4CG is the simplest calculation for TM though many design programs calculate it automatically Your annealing temperature should be ~26C below TM
47 Design of Primers Preferable if each primer spans an intron This way the primer will largely ignore genomic DNA contamination
Junk (non-coding) genomic DNA Gene Transcription mRNA Reverse Transctiption (RT) cDNA 48 Intron Spanning During PCR, primers will bind to the cDNA made from the mRNA, but not genomic DNA
primer genomic DNA cDNA Finding a primer pair that will do this and everything else can be impossible 49 Is the cDNA for Collagen II present?
cDNA (Collagen II) cDNA (GAPDh) cDNA (EGF receptor) cDNA (TGFalpha) cDNA (Collagen II) cDNA (GAPDh) cDNA (EGF receptor) cDNA (TGFalpha) 50 Primer check It's easy to be sure that your primer will stick to your cDNA of interest (as long as you know its sequence) Will your primer stick to any other cDNAs that might be in your sample, though? 51 BLAST 52 Input your primers here, with a blank line between them Choose "others" to look at all databases or target your choice to a particular species 53 Expect a longer wait when the server is busy 54 Intro graphic: Not necessary for primer design. Better suited to finding homologous sequences in the target genome 55 Best matches on top Often there are multiple records for the same protein The statistics of sequence similarity scores
56 Shows where the each primer binds on the cDNA (which gives you the expected product size!) 57 Analysis of Data Run RT-PCR products on an agarose gel (with a DNA stain) Visualize with UV/near UV light Question: if you add different amounts of total RNA to your samples, how can you compare them? 58 Easy mistakes to make When your volume is 1 L, one bubble = no reagent Use only RNase-free (or sterile) plastic and pipet tips with filters Keep all reagents on ice and capped 59 Wastes and Hazards 8a The RNeasy kit components (which all go into the same waste bottle) have various hazards: Buffer RLT contains guanidine thiocyanate: irritant, toxic Buffer RW1 contains ethanol: flammable Buffer RPE contains guanidine hydrochloride: irritant 8b Mostly harmless
View Full Document
This note was uploaded on 10/14/2011 for the course BIOE 115 taught by Professor Haley during the Spring '11 term at City College of San Francisco.
- Spring '11
- Tissue Engineering