* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download PCR settings, pitfalls and artefacts
Board on Science, Technology, and Economic Policy wikipedia , lookup
Gene therapy wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Gene nomenclature wikipedia , lookup
Gene expression profiling wikipedia , lookup
Long non-coding RNA wikipedia , lookup
RNA interference wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Designer baby wikipedia , lookup
Gene prediction wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
SNP genotyping wikipedia , lookup
RNA polymerase II holoenzyme wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
Epitranscriptome wikipedia , lookup
Gene regulatory network wikipedia , lookup
RNA silencing wikipedia , lookup
Non-coding RNA wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
De gekoppelde afbeelding kan niet worden weergegeven. Het bestand is mogelijk verplaatst, heeft een andere naam gekregen of is verwijderd. Controleer of de koppeling verwijst naar het juiste bestand en de juiste locatie. PCR settings, pitfalls and artefacts Dr. Sci. Sabine Franke May 17th 2013 Molecular Biology and Cytometry Course SCK CEN - Mol Overview • Qualitative PCR Reagents Primers Thermocyclers Controls Nested PCR • Quantitative PCR Qualitative PCR PCR optimization • sufficient and specific amplification. Problem • Low yield or no amplification product • Multiple, nonspecific amplification products Check basic parameters • • • • • • • • • • • • template quality template quantity PCR primer design reverse transcription magnesium concentration cycling parameters reaction buffer composition enzyme concentration cycling parameters PCR additives Thermocyclers others Template quality • RNA quality and purity is crucial for success • Extraction and storage of RNA important Template quantity • Avoid differences in RNA quantity PCR primer design • Length ranging from 15–30 bases • G+C content 40–60% • Avoid internal secondary structure (primer dimers/ hairpins) • Avoid three G or C nucleotides in a row near the 3´-end • Ideally, both primers should anneal at the same temperature. Annealing temperature starting ~ 5°C below calculated Tm • Purity of primers (desalted, HPLC,…) PCR primer design Nonspecific priming - Secondary products and lack of specificity Primer dimer formation – Lower yields Lower sensitivity IDT,Eurogentec, Promega,…. BLAST search; eventually other primer Reverse transcription Different reverse transcriptases have significantly different efficiencies. Reverse transcription One step RT One step RT PCR is performed sequentially in the same tube using the entire amount of cDNA synthesis products as the PCR template. Two step RT Two step RT-PCR are performed sequentially, but only a portion of the cDNA products is used as the template for PCR, which is performed in a separate tube. Reverse transcription • One or two step RT-PCR One-step RT-PCR more sensitive and requires less pipetting Two-step RT-PCR allows multiple PCRs from a single RT reaction to quantify multiple targets or perform replicate assays. Reverse transcription • amount of RNA • RNA quality and purity can affect first-strand cDNA synthesis efficiency RNA degradation -> efficientcy RT reaction Magnesium concentration • Effects of magnesium concentration on PCR amplification Magnesium is important cofactor to help stabilization of DNA structure. Cycling parameters Number of cycles: 25-40 cycles Reaction buffer composition Choise of nucleotides Enzyme concentration • Concentration influences PCR reaction PCR additives Has to be checked for each protocol (example DMSO) Thermocyclers • Calibration - thermocyclers can loose calibration over time • Some blocks on thermocyclers do not support “fast cycling” • Older thermocyclers need mineral oil layer over reactions Others • DNA contaminants (labo area) • plastics Problem: No Product Thermocycler Issue X [MgCl2] XX Template/ Primer X Cycling Conditions X Enzyme X Reagents XX Cloning Issues XX XX Wrong Sequence Non-Specific XX XX X XX XX Troubleshooting Weak or no amplification: increase # cycles increase time at step(s) increase quantity of template increase Taq concentration by 2X try different Taq (e.g. Faststart) increase MgCl2, primers, dNTPs Smears above band decrease # cycles decrease concentration of reagent(s) Sensitivity problem Certified reference material cell line documentation and verification Troubleshooting low weight bands: decrease MgCl2 concentration decrease # cycles raise annealing temperature decrease primer concentration decrease dNTPs high weight bands decrease Taq concentration Prevent How? Bad sequence Perform a bioinformatic evaluation Contamination separate RNA area sample prep, mix set-up, PCR flow Contamination Add DNA or RNA to your wells last cross contamination filter tips (aerosol-resistant) freeze/thaw cycles Make aliquots Bubbles Centrifuge PCR plate before loading Controls Control requests • Plasmids work as control templates but can have slightly different conditions compared to gDNA or cDNA • If possible: Certified reference material (cell line documentation) • negative control(s): check for contamination • Run a positive control (a sample known to amplify well) Control • Include an internal standard in your PCRs • second primer pair that amplifies a “housekeeping” gene can be included in the reaction • Participating in external QC rounds Nested PCR Nested PCR Attention • Special care for nested PCR! More sensitive, but … • opening increases the risk of contaminating subsequent reactions with amplified product. • Never opening more than one tube at a time • Using a separate thermocycler • Adding additional negative control Quantitative PCR De gekoppelde afbeelding kan niet worden weergegeven. Het bestand is mogelijk verplaatst, heeft een andere naam gekregen of is verwijderd. Controleer of de koppeling verwijst naar het juiste bestand en de juiste locatie. Quantitatif PCR design Quantif. samples analysis assays guidelines Ref.gene Quantitative PCR • Include an internal standard in RT-PCRs. “housekeeping” gene • Housekeeping genes can help account for differences in the amounts of starting nucleic acid. Tips Change positions of controls Tips Pipette calibration frequently Tips • Do RNA extraction in one serie • Do RT step in one serie Tips Participating in QC rounds not only for tests but also for extraction/storage conditions Housekeeping gene • Include an internal standard in RT-PCRs. For example, a second primer pair that amplifies a “housekeeping” gene can be included in the reaction. (constant expression levels among the samples compared) Housekeeping gene • Stable expression • Should be expressed in all cells • HK gene should have the same copy number in all cells • Transcription level in same range as target gene Perfect standard does not exist Gene expression Gene expression result: impact of RNA quality Accuracy of gene expression is highly dependent onmRNA quality Reference gene expression stability is influenced by RNA quality Quantitative RT-PCR partially degraded RNA may not give accurate representation of gene expression Standard-curve method Absolute and relative quantification Interpretation/quantification outside standard curve Efficienty • Control the efficientcy of the reaction Variables which can affect the efficiency: - Length of the amplicon - Secondary structure - Primer design Conclusion • check working area before starting • check primers before starting • check protocol before starting • check settings (cycles, baseline, threshold) Before starting • use controls • analyse problems step for step Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention Thank you for your attention