Why Analyse RNA ? • Transcriptional Profiling • Levels of mRNA expression • mRNA: early step in gene expression • Controlled step • Variation between different cases – Normal Vs Disease – Responders Vs Non-Responders
ANALYSIS OF GENE EXPRESSION 5’ DNA (gene): 3’ PROMOTER exon 1 intron exon 2 transcription 3’ 5’ RNA (1 o Transcript) RNA processing Polyadenylated 5’ 3’ RNA analysis mRNA m 7 GpppN AAAAAA translation Note: Non coding Introns are not protein included in mRNA molecule
RNA Analysis RNA RT-PCR Microarray Analysis Quantitative PCR •Comparison of mRNA expression profiles between two states; •Disease Vs Normal •Treated Vs Untreated •Primary Vs Mets
Working with RNA RNA is more susceptible to degradation than DNA The 2´ hydroxyl groups adjacent to the phosphodiester linkages in RNA are able to act as intramolecular nucleophiles in both base- and enzyme-catalysed hydrolysis. DNases require metal ions for activity and so can be inactivated with chelating agents e.g. EDTA RNases bypass the need for metal ions by taking advantage of the 2´ hydroxyl group as a reactive species .
Problems with RNases • RNases – single-strand specific endoribonucleases – resistant to metal chelating agents – can survive prolonged boiling or autoclaving • But… – relies on active site histidine residues for activity – Therefore, it can be inactivated by the histidine- specific alkylating agent diethyl pyrocarbonate (DEPC).
Avoiding ribonucleases Exogenous Introduced during working procedures Eliminate through good working practices Endogenous Released by cells or tissue during extraction Eliminate through use of inhibitors of RNase activity
Working with RNA – Dos and Don’ts Always wear gloves - Skin is an abundant source of ribonucleases. Prepare solutions for RNA work using autoclaved glassware, then autoclave the solutions after they are prepared. Better still use disposable plastic ware if possible. If possible use pre-sterilized water. Use separate solutions for RNA work and only use them for RNA. DEPC treatment of water isn’t always necessary. Autoclaving water and solutions can sometimes be more effective in removing RNases than chemical treatment.
Working with RNA – Dos and Don’ts If you do need to treat your solutions with DEPC: 1.make your solution 0.1% DEPC (500 µl in 500 ml H 2 O) 2.shake it well 3.keep it overnight at RT 4.autoclave Take care! DEPC is highly carcinogenic. Use a fumehood!
Working with RNA – Dos and Don’ts Maintain a separate area for RNA work that has its own set of pipettes. This is especially important if your work requires the use of RNase A (e.g. plasmid preps). Sterile, disposable plasticware can safely be considered RNase-free and should be used when possible Use RNase away or RNase zap!!
RNA Extraction …an example
TRI Reagent TM • Sigma (Cat# T9424) • RNA, DNA and Protein extraction • Cell/Tissue lysis • Liquid separation • Quick and Effective
Extraction from Cells in Culture • 80 – 100% Confluent T75 (~1 x 10 7 cells) • Remove all media, wash twice with PBS (saline) • Add 1ml TRI REAGENT (cover all cells) – Scale Down/Up for other culture vessels • 10 min at Room Temp (RT) • Remove to sterile microfuge tube
Extraction from Tissue • Remove tissue from RNAlater into sterile microfuge tube. • Add 1 mL TRI REAGENT • Homogenise at 15,000 rpm for 1-2 min. • Wash Tip between samples in 100% Ethanol, then 0.1 % DEPC.
Chloroform Separation • Add 200 µL (0.2 ml) Chloroform (fumehood!) • Mix well (vortex), and stand at RT for 15 min • Centrifuge at 12,000 x g (MAX!) for 15 min at 4 o C • 3 layers: – Upper (aqueous): RNA – Middle (interphase): Protein – Lower (organic): DNA • Remove upper phase to fresh microfuge tube.
Propanol Precipitation • Add 500 µL Ice-Cold Isopropanol and mix • Stand on Ice for 10 min • Centrifuge at 12,000 x g for 10 min at 4 o C • Pellet ? • Remove Isopropanol • Add 1 ml 75% Ethanol and vortex • Centrifuge at 7,500 x g for 5 min • Remove Ethanol and allow to air dry (10 min) • Resuspend in 10 – 50 µL 0.1% DEPC (60 o C 10 min)
RNA Quantitation
RNA Quantitation • UV Spectroscopy • 1/100 dilution of RNA – 5 µL RNA in 495 µL 0.1 % DEPC • Absorbance at 260nm and 280nm – Quartz cuvette – Blank with 0.1% DEPC
RNA Quantitation Calculation • A 260 = 1.0 (40 µg/mL) • Concentration (µg/µL) = A 260 x 40 x 100 (diln. factor) 1000 (mL → µL) • Or Simply: A 260 x 4 = Concentration (µg/µL) • A 260 /A 280 Ratio: RNA Quality/Purity ( ≈ 1.8) – Higher: Organic Contaminants – Lower: Protein Contaminants
RNA Quantitation Calculation • Eg – 1/100 dilution of RNA – Absorbance Values: • 260nm 0.456 • 280nm 0.250 – Concentration: • (0.456 x 40 x 100)/1000 • 0.456 x 4 = 1.824 µg/µl – Purity • 0.456/0.250 = 1.8 (perfect!) Newer technologies : BioAnalyzer NanoDrop
Next? Assessment of RNA Quality
Agarose Gel Electrophoresis • To assess Quality of RNA – Extent of degradation • Also used to as standard method for analysing, identifying and purifying fragments of DNA (later).
“Electrophoresis” • A technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids – based on their difference in size, charge or V I conformation. • When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. • In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.
“Electrophoresis” • Nucleic acids are electrophoresed within a matrix or "gel". • The gel is cast in the shape of a thin slab, with wells for loading the sample. • Agarose is typically used at concentrations of 0.5 to 2%. • The higher the agarose concentration the "stiffer" the gel. • Agarose gels are extremely easy to prepare: simply mix agarose powder with buffer solution (TAE/TBE), melt it by heating, and pour the gel. It is also non-toxic. • The gel is immersed within an electrophoresis buffer (same as above) that provides ions to carry a current and it also maintains the pH at a relatively constant value.
RNA Electrophoresis 1. The agarose gel with three slots (S). 2. Injection of RNA sample into the first slot. 3. Injection of samples into the second and third slot. 4. A current is applied. The RNA moves toward the positive anode due to the negative charges on its phosphate backbone.
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Gel System
Procedure • Clean Gel System with RNase Inhibitor Spray • Mix 50ml 10X TAE (Tris Acetate EDTA) buffer with 450ml DIW (De-ionised Water) = 1X TAE • 0.5g Agarose in 50ml 1X TAE Buffer – 1% (w/v) solution/gel – Microwave until dissolved (1-2min @ 650W) • Pour into casting tray (with combs) and allow to cool/solidify
Procedure • Analyse 2µg RNA by electrophoresis – 2/concentration (µg/µl) – Eg: • 1.824 µg/µl • 2 µg in ~ 1 µl • Mix with 1 µl DEPC and 0.5 µl RNA loading buffer • Heat 65 o C 10 min then chill on ice • Submerge gel in 1X TAE (running buffer) • Load RNA (2.5 µl) on gel and run at 100V
Procedure • Remove gel after ~ 40min (blue of buffer almost at end of gel) • Visualise under UV light • Visible ribosomal subunits indicate intact RNA – 1 Degraded – 2,3 Good Quality
RT-PCR Reverse Transcriptase Polymerase Chain Reaction
The basics… • Interested in gene expression (mRNA) • Levels of mRNA (transcripts) • Comparison between 2 states (normal and disease) • mRNA (1-5% of total RNA) • We use PCR (DNA Technique) – More on that later • Must Convert RNA to DNA – How ?
Reverse Transcription • mRNA molecule is copied into a double stranded DNA compliment (cDNA) • Reverse transcriptase – enzyme that performs this. • Used naturally by retroviruses to insert themselves into an infected organism's DNA genome • cDNA contain coding regions only (exons)
Reverse Transcription (RT) • mRNA Template • ‘Priming’ – polyA mRNA isolated from total RNA using oligo dT primer • Polynucleotide of T’s – Initiates synthesis • First strand of cDNA synthesised using Reverse Transcriptase (RT) enzyme – Adds complimentary nucleotide bases to mRNA to make cDNA
cDNA is then used as a template in PCR
PCR • Polymerase Chain Reaction – Technique for Targeted DNA Amplification – Starting material ('target sequence’); • A gene or segment of DNA (cDNA in our case) – Target sequence can be amplified a billion fold in a matter of hours • PCR allows one to take a specimen of genetic material, even from just one cell, copy its genetic sequence over and over, and generate a test sample sufficient to detect the presence or absence of a specific virus, bacterium or any particular sequence of genetic material
PCR Applications • Widely used in molecular biology • Specific Amplification • Assuming sequence of target is known; – Viral Detection • HIV can be quantitated – Screening genes for mutations – Detecting gene expression – Detection of food pathogens – Forensic identification
The PCR Reaction • Template – Target DNA that Primers will bind • Primers – Bind target sequence, making the reaction specific • Taq – enzyme which carries out the amplification reaction – extends the primers from their binding-sites on the target along the template • Buffer – Contains a salt (KCl) and MgCl (cofactor for Taq) • Nucleotides – A,T,G and C – Deoxyribonucleotide triphosphates (dNTPs) – DNA building blocks • Water – High ‘PCR’ grade
PCR: Practicalities • Always wear gloves • All reagents must be thawed and mixed completely before use • Typical Reaction Mix (50µl); – 37.5µl sterile water – 5µl 10X Buffer – 1µl 10mM dNTP mix – 0.5µl Taq (5U/µl stock) – 1µl Primer 1 & 1µl Primer 2 (10 pmol/ul) – 5µl Template (cDNA)
The PCR Procedure • Entire genomic double stranded DNA is heated (denatured) • Primers (DNA oligonucleotides) – flank the nucleotide sequence of the gene – synthesised chemically – Prime DNA synthesis on single stranded DNA • In vitro DNA Synthesis catalysed by DNA polymerase • Primers remain at 5’ end of new DNA fragments
The PCR Cycle • Initial Cycle: 1min @ 95oC • Followed by 40 cycles of following; – Denature: 1min @ 95 o C – Anneal: 1min @ 50-60 o C (depends on primer) – Elongate: 1min @ 72 o C • Final extension: 10min @ 72 o C
PCR Links • Calculate Annealing Temperature – http://www.bioinformatics.vg/bioinformatics_to ols/oligo2002.shtml • Primer Design – http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi – http :// www.basic.nwu.edu/biotools/oligocalc.ht ml
The Thermocycle
The PCR Procedure
PCR: DNA Amplification
Agarose Gel Electrophoresis • Analysis of PCR product • Mix 50ml 10X TAE (Tris Acetate EDTA) buffer with 450ml DIW (De-ionised Water) = 1X TAE • 0.5g Agarose in 50ml 1X TAE Buffer – 1% (w/v) solution/gel – Microwave until dissolved (1-2min @ 650W) • Add 1µl 10mg/ml Ethidium Bromide and mix – Interacts with Nucleic Acids – Fluorescent Complex – Visible under UV • Potent Mutagen – Fumehood, Lab Coat, Safety Glasses, Gloves – Spills: Absorbed and Decontaminated with soap and water • Pour into casting tray (with combs) and allow to cool/solidify
Running the Gel • Submerge gel in 1X TAE (running buffer) • Mix 3µl PCR reaction with 3µl loading buffer and load onto gel • Mix 3µl 100bp DNA ladder with 3µl loading buffer and load onto gel • Run at 100V • Remove gel after ~ 40min (blue of buffer almost at end of gel) • Visualise under UV light • Dispose Gel in Yellow Biohazard Bin
DNA Electrophoresis 1. The agarose gel with three slots (S). 2. Pipette DNA ladder into the first slot. 3. DNA ladder loaded. loading of samples into the second and third slot. 4. A current is applied. The DNA moves toward the positive anode due to the negative charges on its phosphate backbone. 5. Small DNA strands move fast, large DNA strands move slowly through the gel. DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel. 6. The DNA is spread over the whole gel. The electrophoresis process is finished.
Real Time PCR
Traditional PCR – Limitations • Qualitative not Quantitative • Gel Required • End point detection • 4/5 hrs until result • Non numerical • Non Automated
Real Time PCR • Monitor Amplification in Real Time • Measure the kinetics of the reaction in the early phases of PCR • Quantitative and Qualitative • 30/40 min until result • Numerical output • Automated
PCR Phases Reaction components being consumed. Reaction is slowing. Doubling of product at Reaction has stopped. every cycle. No more products are being made. Area for Real Time Detection
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