Fundamentals of RT-qPCR and Gene Expression NOTES PDF

Highlighted text - ni ask si sir dex about ani Course 1: Introduction to RT-qPCR and Gene Expression Chapter 1: Qualitative vs Quantitative Analysis Polymerase Chain Reaction Polymerase Chain Reaction (PCR) → Kary Mullis (1983) → cornerstone of molecular biology → targets specifi...

Highlighted text - ni ask si sir dex about ani Course 1: Introduction to RT-qPCR and Gene Expression Chapter 1: Qualitative vs Quantitative Analysis Polymerase Chain Reaction Polymerase Chain Reaction (PCR) → Kary Mullis (1983) → cornerstone of molecular biology → targets specific molecular interest within the samples Qualitative PCR: tests to see if the specific target of interest is present or absent in the samples - Might use a conventional thermal cycler to amplify up their DNA template - Uses a DNA gel in the presence of a DNA specific marker to visualize the presence or absence of their target; target DNA is visualized as a band Quantitative PCR: tests how much of that target of interest is present - Can be classified as either absolute or relative - Absolute: standard curve is used to derive the concentration of an unknown sample ; a standard curve using a sample of known concentration (which is serially diluted) is run alongside the sample of unknown conc. ; useful for quantifying bacteria or viruses - Output: an absolute or exact measurement (e.g. actual # of copies of the target of interest present in the sample) - Relative: more useful and generally easier ; applies universally regardless of sample input ; most commonly used for gene expression ; standard curve is not required - Output: relative terms e.g. gene expression was upregulated 32-fold in response to treatment Which of the following applications would be best tested with a quantitative analysis? a. Presence or absence of a DNA target b. Viral load quantification c. Gene expression d. GMO detection Answer: B and C - Options one and four are qualitative analysis. Viral load quantification requires quantitative analysis (as its name suggests). Gene expression to compare levels of transcripts between different samples also requires quantitative analysis. Chapter 2: DNA Structure and Reagent Considerations for DNA Amplification Deoxyribonucleic acid (DNA) → made up of 4 different subunits called bases: - Purines: pyrimidine ring fused with an imidazole ring – Adenine (A) and Guanine (G) - Pyrimidines: single pyrimidine ring; structurally smaller than purines – Cytosine (C) and Thymine (T); T is replaced with Uracil (U) in RNA → bases (on a single strand) are linked together by peptide bonds on a sugar-phosphate backbone → two strands that make up double stranded DNA are described as complementary - A–T: 2 hydrogen bonds - C–G: 3 hydrogen bonds → nitrogenous base is attached to 1’ of sugar ring, phosphate group attached to 5’, and exposed hydroxyl group attached to 3’  → during amplification, additional bases are connected by linking the 5’ carbon of one pentose ring to the 3’ carbon of the growing nucleotide chain via a phosphate group, forming a phosphodiester linkage → exhibits directionality; DNA polymerase moves along the template strand in a 3’ to 5’ direction; daughter strand is generated in a 5’ to 3’ direction - PCR amplification requires: right reagent & right temperature conditions - Core reagents: amplification supermix, PCR primers specific to target of interest, sample (also called template), PCR grade water - Supermix: key to success; sometimes referred to as mastermix (supermix + primers + probes + water, prior to adding the sample) - dNTPs: single base building blocks used to generate new DNA molecules during amplification - Buffers: required to maintain a favorable environment during amplification - DNA polymerase: PCR enzyme powerhouses that bind at the priming sites and incorporate free-floating dNTPs accdg. to template strand ; modified DNA polymerase may be used to difficult samples (e.g. long amplicon or a sample high in amplification inhibitors), ensuring a more stable interaction and increasing amplification efficiency - Primer: short (around 18-24 base oligonucleotides) and come in pairs (called forward and reverse for each strand of DNA) ; bind to the DNA template, creating an attachment site for the DNA polymerase, and act as an initiation site for amplification to take place ; essential to bind only to the specific target of interest; consider specificity of the sequence, primer length, base balance and distribution - Template: sample of interest - Water: should be RNAse and DNAse free to avoid degradation of the starting template ; used to bring all the reactions to an appropriate and consistent final volume Which of the following base pairings are held together the strongest in DNA? a. Adenine - Thymine b. Adenine - Guanine c. Cytosine - Guanine d. Adenine - Uracil e. All are held together with equal strength Answer: C - Cytosine and Guanine are held together by three hydrogen bonds, while adenine and thymine are held together by two hydrogen bonds. Uracil is not typically found in DNA. Chapter 3: Temperature Considerations for DNA Amplification DNA Amplification - Denature (Denaturation): dsDNA or cDNA is heated up to 95C ; hydrogen bonds between cDNA bases break or melt, resulting in 2 complementary ssDNA template strands - Anneal (Primer annealing): reaction is cooled down to 55-65C ; exact temperature chosen should be the optimal temperature for the specific primer pair to attach to their intended specific location on the DNA template, with forward and reverse primers separate end-to-end by around 100 bases ; too cool temperatures lead to primer-dimer and hairpin formation ; too high temperatures interfere with primer binding - Extend (Extension): DNA polymerase enzyme attaches at the priming site and moves along the original template strand in a 3’ to 5’ direction ; incorporates free-floating dNTPs to the 3’ end of the new complementary strand ; resulting product after a single cycle of PCR is 2 complete dsDNA  ; TAQ DNA Polymerase - extracted from Thermus aquaticus; extremely thermotolerant hence it is used to attach the nucleotides to the growing DNA strand → Process above is repeated typically for a total of 40 cycles → 2-step protocol: denature, combined primer annealing and extension - Used for standard length amplicons (100bp or less) with 30 second combined primer anneal/extension step → 3-step protocol: denature, primer annealing, extension - Extension temperature is set to the optimal temp for polymerase activity, typically around 72C - Used for long amplicons (>150bp), complex samples (PCR inhibitors), low efficiency amplification * In theory, after each cycle of amplification, no. of copies of the target doubles - Around 1x1012 copies after 40 cycles - Does not apply in real life as reaction conditions become less favorable during the later cycles and the efficiency of amplification decreases - Caused by finite dNTPs, buffering instability, and unfavorable reaction dynamics Your target sequence is 165 base pairs long and the efficiency of amplification is low. You might want to… a. Increase your denaturing temperature to 97 degrees to allow more complete strand separation b. Increase your number of amplification cycles to 45 to allow for more complete amplification c. Use a 3-step instead of a 2-step amplification d. Run a temperature gradient to better optimize your assay e. Increase your sample input concentration to reduce the relative Cq Answer: C and D - Because 165 base pairs is a long target, using a 3-step process with a dedicated extension step will allow the polymerase to work at its optimal temperature. Additionally, running a temperature gradient will ensure you are using the best possible annealing temperature, and getting the best possible amplification efficiency. Chapter 4: PCR Detection Chemistries SYBR Green PCR Detection: - Conventional PCR (for qualitative analysis): run to end-point; all amplification cycles are performed prior to nucleic acid detection using DNA gel and DNA specific marker - Real-Time PCR: nucleic acid detection is performed on-board the real-time PCR instrument after each of the 40 PCR cycles - Incorporates fluorescent DNA specific markers to make DNA visible - Fluorescence is absorbed by the DNA specific fluorescent marker inside the reaction which then re-emits the energy at a different wavelength that is detected and recorded by the real-time system Fluorescent chemistries: - Dye-based chemistry: e.g. SYBR green - Supermix: dNTPs, polymerase, buffers, SYBR green - SYBR green: intercolating agent that binds to the minor groove of dsDNA ; binds indiscriminately to any dsDNA products ; must verify no non-specific amplification nor secondary structures (hairpins – unpaired loop of mRNA that is created when an mRNA strand folds and forms base pairs with another section of itself, primer dimers – self-annealing of primers) - Primers - Template - Water - Probe-based chemistry Which of the following can result in off-target signal in your SYBR Green RT-qPCR experiment? a. Secondary structures like hairpins b. Running your reaction too hot during primer annealing c. Primer dimers d. Pseudogenes  e. Running a melt curve after amplification completion Answer: A, C and D - SYBR Green binds indiscriminately to any double-stranded regions. Therefore, any double-stranded regions that exist outside the desired gene such as hairpins, primer dimers and pseudogenes may also bind to SYBR Green, leading to an increase in off-target signal. Chapter 5: PCR Detection Chemistries Probes → Probe-based chemistry: probe oligonucleotides are designed in conjunction with the assay specific primers to be specific to the target region of interest - Hydrolysis probe: most common type of probe - Probe must sit in between the forward and reverse primer, but outside the primer binding region; achieved using a short (~20-30 base) sequence-specific oligonucleotide complementary to the target sequence - Oligo is conjugated at the 5’ end with a fluorescent reporter dye (e.g. FAM, HEX) - 3’ end is conjugated with a quencher molecule - When probe is not bound to the DNA template, fluorescence is not detected as the energy absorbed by the fluorescent reporter is not freely emitted, but is absorbed by the closely located quencher where it can be emitted in a wavelength (not detected by real-time system) or as heat; this process is called FRET - Forster Resonance Energy Transfer - When the probe is hydrolyzed by the polymerase, the reporter breaks free from the quencher; fluorescence is released and detected - Probe attaches first to the DNA strand before the primers during the annealing phase - It is possible to detect multiple different targets of interest (multiplex) by combining probes with different reporter fluorophores Real-Time PCR Protocols - Always look at the specific product information sheet - Both probe-base and dye-based chemistries start with a hot-start step - 95C incubation step where the polymerase enzyme is activated - Initial denaturation of DNA template occurs - Cycling phase: denaturation, annealing, extension - Melt-Curve: only for dye-based chemistry; used to verify amplification specificity, 95C where 0.5C/5 secs - Melting point: temperature at which 50% of the DNA is denatured; substances have different melting points PROS: SYBR Green: inexpensive, can run melt-curve Probes: multiplex capability, increased specificity CONS: SYBR Green: can’t multiplex; not tolerant to non-specific amplification Probes: need to run gels to confirm specificity, more expensive, more complex assay design Probe-based chemistry is a better option than using SYBR-Green when… a. Cost is your biggest deciding factor b. You want to multiplex multiple targets per well c. You have multiple similar targets d. Your sequence is greater than 80 bases in length Answer: A, B and C - There can be multiple reasons to run probe-based chemistry. You need to run probe-based chemistry when you want to multiplex multiple targets per well. This is something that cannot be done with SYBR Green. Probe-based chemistry is also a better option when you have multiple similar targets because your primer and probe has to be specific. Budget may also be a factor in your selection. Chapter 6: Getting Started With Amplification Curves Core processes of RT-qPCR: amplification and detection Data output: - Level of fluorescence is measured and recorded for each experimental reaction well - Initial cycles of amplification: low fluorescence, low-level background fluorescence - “Take off”: point that the level of fluorescence positively deviates from the background fluorescence - Amplification curve: line through each of the data points; one amplification curve per assay - Exponential phase - Non-Exponential / Plateau Phase Structure of the Amplification curve: > Baseline: amplification begins, flat line in linear view, fluorescence too low to be detected > Take Off: amplification takes off, abundance of PCR reagents, exponential increase, threshold should be set in this phase, best amplification efficiency and precision > Linear: amplification decreases, PCR agents decreasing, decreasing amplification efficiency and precision, linear increase > Plateau: amplification flattens off, depletion of PCR reagents, self annealing of PCR products, low/no additional amplification ** not advised to go beyond 40 cycles of amplification as it increases the likelihood of observing non-specific amplification - Quantification cycle (Cq) value: the cycle number at which the threshold line crosses through the amplification curve ; sometimes referred to as the threshold cycle (Ct value) ; can be used to quantify changes in gene expression When setting the threshold line on your amplification curve, you should… a. Set it as high as possible in the linear view (avoiding the plateau phase) b. Always use the software default c. Set it in the region of exponential amplification d. Use the same threshold for all targets e. Use the same threshold for all samples of a specific target Answer: C and E - You want to set the threshold in the region of exponential amplification, so that you can establish the Cq value for each sample. All samples of a specific target should have the same threshold so that you can compare Cq values to determine gene expression changes. Different targets however can have different thresholds as these Cq values are not directly compared. Course 2: RNA Sample Preparation Considerations Chapter 1: RNA Function, Diversity, and Key Characteristics RT-qPCR → highly effective and popular method used in many fields of scientific research → allows scientists to get a closer look at how certain variables affect the synthesis of gene products → is a method that aims at identifying targets within the transcriptome: is this transcript present and if so, how much of it is present in comparison to other targets or other cells of interest? Gene products: determine the function and therefore the purpose that a cell carries out ; Fluid in their makeup depending on environmental cues BASIC SCIENTIFIC DOGMA OF BIOLOGY: illustrates how a cell interacts with its environment to adapt and survive > Genome: packaging of genes within a cell’s nucleus > Transcription: a process by which a cell synthesizes different transcripts from genes ; transcripts comprise important information used by the cell as a form of transcriptional and translational regulation  > Transcriptome: a milieu of different transcripts; uncovers more on how a cell is functioning which is important in pathology > Translation: protein synthesis from a transcript > Proteome: complete set of proteins expressed by an organism; assortment of proteins produced at a specific time in a particular cell or tissue type Transcriptome: - 80% of genome is actively transcribed into the transcriptome in mammalian cells - Roughly 90% is assigned to the general RNA biotype termed non-coding RNA (ncRNA) → RNA transcripts that do not mature into proteins yet play pivotal roles in mediation transcription and translation → e.g. small nuclear RNAs, short hairpin RNAs, transfer RNAs, ribosomal RNAs - Ribosomal RNAs make up the majority of transcripts within the transcriptome due to their large functional requirement during translation - Quantifying ribosomal RNAs is used as a positive control during RNA extraction - Coding RNA: 2-5% of the transcriptome; describes transcripts that contain a code or message used during protein synthesis, therefore these transcripts are also collectively known as messenger RNA or mRNA - mRNA: predicts the proteome makeup and the current function of the cell; protein-synthesis potential Transcription: process where DNA is transcribed into RNA that will be used for protein synthesis → Step 1: The first step includes the unwinding of DNA using the RNA polymerase to promote access of the transcription machinery to the segment to be transcribed. → Step 2: The RNA polymerase and associated transcription factors recognize these unwound genes whereby RNA polymerase facilitates the pairing of complementary nucleic acids to form an RNA copy of the sequence, producing a slightly different combination compared to the DNA strand. Thymine (T) is replaced by Uracil (U) in the RNA. → Step 3: The newly synthesized single stranded transcript will undergo a process that turns the pre-mRNA (precursor mature RNA or heterogeneous nuclear RNA) into a mature mRNA via RNA splicing. In this process, the introns (non-coding part of the RNA) are cleaved. There is an addition of a 5’ guanine cap and a 3’ poly adenosine tail, which are used for stabilization and protection of the mature mRNA molecule as it proceeds to the ribosome for translation. - ncRNA regulate gene expression, translation and transcription → Step 4: The mature mRNA leaves the nucleus. Translation: involves both rRNA and tRNA to facilitate the ribozyme in efficient protein synthesis → Step 1: The mature mRNA will be transported to the ribosome to proceed with protein synthesis. This involves both ribosomal RNA and transfer RNA. - The mRNA is not directly involved in the process; tRNA is required for this. → Step 2: Each three-base stretch of mRNA is known as a codon, and each codon corresponds to a specific amino acid. As the mRNA passes through the ribosome, the codons interact with the anticodon of the tRNA molecule which carries the specific amino acid to include in the growing protein chain. The tRNA will then leave the ribosome. → Step 3: This will continue until the stop codon is reached. → Step 4: A single chain protein (called a polypeptide chain or primary protein) is synthesized. RNA Biotype Characteristics: - Coding RNA: (e.g. mRNA) - Contains a coding sequence (exons) that is used during translation - 5’ cap and 3’ poly-a tail (untranslated regions) which provides more stability as it continues towards the translation phase - Short half-life which requires careful handling in the lab - Non-coding RNA: (e.g. tRNA) - Diverse group of RNA transcripts which do not contain a coding region - Range in size from 19 nucleotides - 100k nucleotides - Most ncRNA do not contain the 5’/3’ processing caps that the RNA contain - Short half-life Which of the following RNA biotypes encode gene proteins? a. miRNA (micro RNA) b. mRNA (messenger RNA) c. rRNA (ribosomal RNA) d. tRNA (transfer RNA) Answer: B - mRNA does contain the code in exon form that is translated into amino acid segments or proteins during translation. Chapter 2: RNA Isolation: The Importance of Pure, Intact Extract RT-qPCR Workflow: Step 1: Isolate RNA - Samples of cells will be used to harvest or isolate the desired collection of RNA biotypes Step 2: cDNA Synthesis - The RNA is used to generate cDNA for downstream amplification steps Step 3: Prepare Real-Time PCR Reaction Step 4: Cycle in Real-Time PCR Instrument Step 5: Analyze Gene Expression Data Step 1: Isolation of RNA - Nucleic acids extracted from biological material need to be free of components which may inhibit enzymatic reactions - Semi-quantitative measurements using RT-qPCR are particularly susceptible to inhibition as small amounts of co-purified inhibitors can have relatively significant effects in downstream analysis → Co-purified molecules (RT-PCR inhibitors): other impurities (may include residual chemicals left-over from the extraction protocol such as phenol from phenol-chloroform), protein, RNA, DNA (can negatively affect PCR amplification) ; proteins and other impurities can decrease the efficiency of two key chemical reactions: Reverse transcription and PCR amplification; will influence the accuracy observed when analyzing RT-qPCR amplitude plots → Reverse transcription: process by which RT-qPCR employs to generate cDNA → PCR amplification: process by which cDNA is amplified to quantify the associated fluorescence of a gene target; assays may bind to the cDNA and the DNA impurity causing an artificial inflated signal - If sample is more purified, Cq values decrease; showing that the purer the sample of RNA, the more accurate and sensitive the experimental assay becomes - Sample integrity: refers to whether the RNA transcripts in the sample are intact or degraded - Reverse transcription and PCR amplification will be inhibited if there are breaks in the transcript of interest - Reverse transcription may not be fully transcribed which may affect PCR amplification - Cq values increase with RNA degradation; showing much lower fluorescent values that what should be reported with intact RNA → kits are often used for RNA extraction mRNA, tRNA, rRNA: use a kit that can perform “total RNA” extraction short-ncRNA/miRNA: seek out specialized short-ncRNA or miRNA kits; avoid column purification steps based on size exclusion → also consider sample type Tissue: prepare RNA isolations with fresh tissue or flash frozen tissue if possible; free thaw cycles will degrade RNA; includes many cell types; represent a reservoir rich in impurities/PCR inhibitors Cells: assess the viability of cells before harvesting; start with the same number of cells per sample; use the recommended no. of cells for the extraction kit Blood: represents a reservoir low in cells/RNA; will need large amounts of blood and good conc. protocol to get enough RNA To keep RNA integrity high and contamination low:  1. Always store RNA samples at -80C 2. Decontaminate all surfaces, pipettors, benchtops, glassware, and gel equipment with RNase-free solutions 3. Wear appropriate PPE, including globes, lab coat, and safety glasses 4. Avoid freeze-thaw cycles to RNA samples 5. Make sure to implement DNAse, especially if working with samples from organisms with very small or no introns. DNase I treatment is the best way to rid an RNA sample from contaminating DNA. (short introns amplify DNA DNA contamination i think) 6. Use aerosol-barrier tips What are two enzymatic reactions impeded by impurities found within an impure RNA extract when performing RT-qPCR? a. Polymerase and the Cell Cycle b. Transcription and Translation c. Reverse Transcription and PCR Amplification d. All of the above Answer: C - Reverse Transcription is the process by which cDNA is synthesized and PCR Amplification is the process by which cDNA is copied over the course of many cycles within the Thermal Cycler. Both reactions can be inhibited by impurities in the RNA extract ultimately affecting the results of our experiment downstream. Chapter 3: Exploring Methods to Validate Extract Quality and Quantity Before starting cDNA synthesis, it’s best to test your RNA extract to define its quantity (primarily determined by the yield of rRNA) and quality (RNA purity and integrity). → Nanodrop: most commonly used instrument to assess RNA quality and quantity; measures the UV absorbance of the various molecules in a sample in a process terms spectrophotometry; uses the Beer-Lambert’s law (absorption is directly proportional to concentration) ; nucleic acids have unique properties that are observed when measuring the UV absorption at specific wavelengths; 230nm (detect contaminants such as phenol), 260nm (concentration of RNA or DNA) and 280nm (potential protein contamination) (both used to measure the ratio of nucleic acids) wavelengths; weighted average of the 260/280nm ratios for the four nucleotides; the average value of RNA should be higher than DNA due to uracil ; 260/280nm ratio: provide insights into the purity of sample regarding whether DNA or other molecules (e.g. protein) is present ; 260/230nm or 260/270nm ratio: used to determine the presence of organic compounds (sugars, urea, salts or phenol) Pros: readily available in most laboratories; enables RNA quantitation with only 1-2μL of sample; wild dynamic range; quick protocol Cons: reading relies upon the purity of samples; contaminating substance with absorption at 260nm or 280nm will affect the estimated OD reading Common contaminants found in UV absorbance, quality assessment 1 and 2 1=proteins and/or phenol (Ex. histones,tryptophan, tyrosine, etc.) 2=EDTA, carbohydrates, and/or alcohols(ethanol, isopropanol, etc.) → Gel Electrophoresis: method of separating molecules, such as nucleic acids, based on the net charge, size and conformation within a matrix ; small aliquots of denatured extract are loaded into microwells within a polyacrylamide or agarose gel ; nucleic acids have an overall negative charge (due to phosphate backbone) therefore they migrate towards the anode ; size of RNA molecules depends on the rate of migration, separating RNA molecules into distinct bands ; bands are stained with EtBr (ethidium bromide) and visualized under UV light  → the use of EtBr is frowned upon because it is a mutagen (cause mutations in DNA), carcinogen, and teratogen (cause birth defects). Alternatives: SYBR Safe, GelRed, GelGreen, and Crystal Violet ; 28S:18S rRNA ratio of intensity to be around 2; deviation suggests RNA degradation ; absence of bads indicates little to no RNA present Pros: cost effective, most labs are equipped with this technology, easy to follow protocol Cons: takes longer than nanodrop, suggested amounts of RNA input can exceed 5μg, qualitative test → Bioanalyzer: performs automated electrophoresis to an RNA sample ; combines microfluidics and a proprietary algorithm to calculate a RNA Integrity Number (RIN) from the intensity ratio (28S:18S) ; higher RIN value reports a higher quality of intact RNA Pros: easily determine the RIN from total RNA sample, uses little input of RNA (~5μl), calculates RNA conc. of high molecular weight RNA biotypes Cons: not always available in the lab space → Qubit Fluorometer: utilizes two unique dyes– one that binds to large, intact and structured RNA and the other selectively binds to small, degraded RNA ; results are presented as a total value for the RNA sample integrity and quality or RNA IQ #, alongside the calculated % of large and small RNA in the sample ; RNA IQ # ranges from 1-10; higher IQ# indicates most of the sample consists of large and structured RNA; smaller IQ# indicates the sample comprises mainly of small RNA with limited tertiary structure Pros: RNA quantity, integrity and quality can be rapidly measured using highly sensitive Qubit assays even in RNA samples with very low conc., most accurate and efficient technique to validate RNA extracts Cons: not always available in the lab space How much RNA is enough to get the results you want? If we assume that the RNA extract is of high quality, the reverse transcriptase and polymerase are performing efficiently, and the quantity of RNA extract is sufficient to satisfy the recommended cDNA synthesis steps within the protocol, then an educated guess can be made based on the abundance of target. - The higher the abundance of gene target, the lower the amount of RNA input is necessary Total RNA: 10pg - 5μg Poly(A) RNA: 10pg - 0.5μg Specific RNA: 0.01pg - 0.5μg - Reproducible Cq values - Logical difference between Cq values of different RNA inputs - Cq values aren’t outside the acceptable range for RT-qPCR detection Picometer (pm): one trillionth of a meter (0.000000000001) Micrometer (μg): one millionth of a meter (0.000001) REVERSE TRANSCRIPTASE AND SARS-COV-2 Reverse Transcriptase is often used in detecting SARS-CoV-2 because the virus's genetic material is RNA, and Reverse Transcriptase is an enzyme that converts RNA into complementary DNA (cDNA). This cDNA can then be amplified and detected using PCR (Polymerase Chain Reaction) techniques. RT-PCR, is highly sensitive and specific, making it ideal for detecting even small amounts of viral RNA in patient samples. In addition, spectrophotometers used to prepare for PCR are usually readily available in most labs, require small amounts of sample to function, and can be performed quickly. Before starting cDNA synthesis, it’s best to test your RNA extract to characterize its… a. Quantity b. Quality c. Density Answer: A and B - Both the quantity and quality of your RNA extract are equally important before moving forward with cDNA synthesis. Chapter 4: cDNA Synthesis: RT Process, Enzymes, and Suitability for RNA Biotypes Step 2: cDNA Synthesis - Two methods available for cDNA synthesis by RT-qPCR: → Two-step reaction: - 2 separated enzymatic reactions: Reverse transcription (RT) and PCR amplification - In RT, primers bind to complementary regions of the mRNA transcript, initiating the recruitment of reverse transcriptase - Reverse transcriptase utilizes the single stranded mRNA molecule as templates to generate cDNA. - During PCR Amplification, primers and probes that are designed to target the specific gene product will bind to complementary regions of cDNA - Polymerase will recognize the bound primers and carry out amplification of the cDNA for 30-40 cycles → One-step reaction: RT and PCR amplification but both enzymatic reactions occur simultaneously in one single tube PROS: One-step reaction: fast (eliminates benchtop work), decreases the chance of contamination (fewer pipetting steps) Two-step reaction: can archive cDNA and use multiple times for additional RT-qPCR rxns, allows for a pre-amp step when targeting rare events CONS: One-step reaction: cannot reuse the cDNA for additional RT-qPCR rxns, conditions in the chemical reaction are not optimal for both RT and Pol Two-step reaction: slower than 1-step, more pipetting steps means increased risk of contamination Properties of RTs: → Not all RT enzymes are created equal - Some have nuclease activity, also known as RNase H+ activity - RTs with the RNase H+ activity has the ability to degrade the transcript as it generates the cDNA, thereby eliminating the transcript from being used in any additional RT reactions Importance of RNase H+ activity: > generates a 1:1 ratio of transcript to cDNA > provides unbiased transcription amid high background > increases likelihood of transcribing rare transcripts (decreases competition for enzyme) > removal of RNA transcripts prevents RNA-blocking of second strand synthesis - E.g. Avian Myeloblastosis Virus (AMV) RT, Moloney Murine Leukemia Virus (MMLV) RT, Human Immunodeficiency Virus (HIV) RT AMV RT: high processivity for reverse transcribing longer transcripts; low RNase H+ activity that amount to lower yields of DNA MMLV (iScript Kits): good thermostability (50C limit); high level of RNase H+ activity that will amount to higher yields; good fidelity/accuracy; best representation and yield HIV: high thermostability (>60C); performs well with GC-rich transcripts; low fidelity/accuracy Priming strategies for generation of cDNA: → Oligo dT which binds to the poly-A tail of mRNA transcripts ; only captures processed mRNA; initiates the synthesis preferentially at the 3’ end of the RNA fragment, which can lead to premature termination if the transcript has increased 2* structure → Random Hexamer primers can also be an option to consider instead of oligo dTs ; these are comprised of random combinations of nucleotides that bind to a variety of potential transcripts in regions outside of the poly-A tail ; can opt for small oligos increases chance of complete coverage; can lead to an overestimation of copy number due to internal priming; may represent a 3’ to 5’ bias → Gene specific consists of primers that anneal to the specific gene product of interest  ; use if you know what your target of interest is; use for 1-step RT-qPCR; best option for the least amount of steps/time ; best for analyzing gene expression ** a mixture of oligos and random primers can often be the best strategy if not using gene specific primers Advantages to performing a 2-step RT-qPCR reaction include: a. The resulting cDNA can be archived and used multiple times for additional RT-qPCR reactions b. Allowing the scientist to perform a pre-amplification step to increase source material (cDNA) from limited sample (RNA extract) c. Accelerated time to results Answer: A and B - The two-step reaction may be advantageous in performing RT-qPCR when your sample source is limited due to the ability to pre amplify the target before performing PCR amplification. Additionally, if the sample that you have harvested RNA from is something that you’d like to use in other RT-qPCR experiments in the future, the two-step method also allows the scientist to store the cDNA in the freezer and use it later if necessary. Course 3: Experimental Design for Gene Expression by RT-qPCR Chapter 1: Defining the Experimental Aim Experimental considerations: - Sample type and treatment - # of biological and technical replicates - Target genes - Reference gene(s) Establish a validated reference gene: (Vondesompele et al., 2002) → When measuring gene expression by qPCR, it is important to use a housekeeping gene or reference gene to normalize the signal between samples. > this accounts for two issues: potential uneven loading of the samples which can occur for multiple reasons, and/or global variability of gene expression. - Amount of starting material must be controlled - Reference gene mRNAs should be stably expressed across sample conditions - Multiple reference genes should be used - Wrote geNorm algorithm to calculate reference gene stability - RNA mass should not be used for normalization purposes → the reference gene reveals either unequal sample loading or global transcriptional noise Pairwise analysis: between samples of different treatment conditions for multiple potential reference genes ; best method for selecting a reference gene ; uses gene selection plates Gene selection plates: contain pre-plated lyophilized primers and would only need to add sample, supermix, and water ; assays are plated in triplicate and duplicated on both sides of the plate ; include several control wells which can be used to verify successful amplification, test for contaminating genomic DNA, verify the quality of RNA and confirm the success of reverse transcription Reference gene stability plot: displays the reference genes in order from best to worst based on its stability value ; stability value was calculated based on the geNorm algorithm ; green - good reference genes; yellow - OK reference genes; red - reference genes to avoid Which of the following are used to refer to the normalizing gene in expression experiments? a. Endogenous control b. Reference gene c. Housekeeping gene  d. Total protein normalization Answer: A, B and C - Reference gene is always needed to normalize gene expression experiments, as noted in the 2009 MIQE Guidelines paper. Historically, endogenous control and housekeeping have been used, they are just not as commonly used today. Total Protein Normalization is used for protein blots, and is a great way to normalize your western blots. Chapter 2: Selecting the Proper Controls Experimental Design – Controls - Needed at the RT and amplification stages Controls: (listed in level of importance) - No template control - No RT control - Positive control - Negative control/ Blank sample - RT positive control - Inter-plate normalizer No template control: most important control; a negative control; always run a NTC for each primer/probe set in every experiment ; reveals contamination or dimers > polymerase > dNTPs > buffers > primers > water ** no template - Must have no amplification; otherwise, it implies contamination - Amplification 10 cycles later than the unknown is still considered acceptable - Melt curve is performed if amplification is observed and see if the Tm is the same with the unknown → melt curve for SYBR assays, gel of PCR products for probes → if the NTC is positive & melt curve is same as sample: - Contamination; can be at lab (e.g. pipettes or reagents) or user level (e.g. aerosolizing template through fast pipetting or accidental spillover) - Decontaminate workspace & pipettes - Prepare fresh working stocks of all reagents - Dispose of common-use water - Include surveillance NTCs with all subsequent runs → if the NTC is positive & melt curve is different from sample: - Possibly primer-dimers - Check complementarity of primer 3’ ends - Run thermal gradient - Titer primer concentration No reverse transcription control: reveals genomic DNA; a negative control > RT Reaction mix > primers > water > RNA **no reverse transcriptase - Must have no amplification → if NRT is positive: - Ensure DNase was used properly - Ensure DNase I is not expired and was stored correctly  - Do PCR primers flank large intron(s)? Positive control: can use samples known to be positive for target of interest to show that mastermix setup and system setup was correct or synthetic templates to determine if samples contain inhibitors ; can be used to test samples for the presence of inhibitors Which are important controls to run in a qPCR experiment? a. No template control b. No reverse transcription control c. No supermix control d. None, maybe I’ll run a control next time Answer: A and B - It is important to run controls in every qPCR experiment. Both the "no template control" and the "no reverse transcription control" are key options to include, to detect whether contamination has occurred. The amplification process cannot happen without a supermix, so it is not recommended to use this as a control. Chapter 3: Biological and Technical Replicates: Best Practices THREE LEVELS OF REPLICATES NEEDED: Technical replicates: variability of the measurement Use technical replicates to: - Measure error of a system (user + instrument) - Ensure another level of quality control for our data - Provide confidence in the measurements and process (today and over time) Biology replicates: variability of the system Cell culture biological replicates: - Clonal cell populations - Replicates should have similar gene expression due to low variability - Fewer biological replicates required to measure variability and for statistical power Organismal biological replicates: - Factors affecting gene expression (age, sex, hormones, genetic variability, circadian rhythm - relates to hormones) - When biological variability is more present, more biological replicates required for statistical power Experiment replicates: overall reproducibility of the experiments Why are replicates needed for gene expression experiments? a. Quantify error in the measurement b. Quantify variability in a population c. Measure the confidence in the measurements Answer: A, B and C - All measurements have error. It may be difficult to know how much error exists unless repeated measurements are made to see how different they are. You will not know how much variation you have between your samples until you measure it. You also won't know how much confidence to have in your measurements until you see what level of variability you have. Chapter 4: Designing Primers and Probes: Best Practices Key assumption of qPCR: template doubles with each cycle of amplification (100% efficiency) Poor primer/probe design leads to: (all lead to poor reaction efficiency) - Amplifying more than one target - Primer dimer formation - Poor binding/ hydrolysis of probe Signs of poor primer design: 1. Poor efficiency, even after optimization - Standard curves measure efficiency  - Plot Cq vs template dilution - Slope between -3.1 and -3.6 are good (90%-110% efficiency) 2. Multiple peaks on melt curve suggest non-specific amplification - Suggests multiple amplicons of different lengths and GC content Rules for designing primer assays: - Keep it short: 60-150bp amplicons → short primer therefore easy annealing - Melting temperature (Tm) usually 55-65C - Deviation of Tm for forward and reverse primer within 4C → Tm of primers must be similar so they work well together - 35-65% GC content → helps with binding due to its stability (3 hydrogen bonds) - No poly-X repeats >4 bases - Avoid self-dimers/heterodimers, hairpins - 3’ GC clamp can improve efficiency → can avoid the self-complimentary at the 3’ end, avoiding primer-dimer formation; helps with the binding of the primer to the template - Intron spanning if possible → differentiates gDNA (can be a contaminant) from cDNA (coding DNA; no introns) Probe design: - Tm 6-8C higher than primers (66-70C) → as the reaction cools down from 95C, the probe anneals first followed by the primer; can be achieved by increasing the length or GC content of the probe relative to the primers - Avoid 5’ G → causes quenching of 5’ reporter - Place probe preferably close to primer - Either strand works for probe annealing - Fluorophores can be swapped (check compatibility) → can use HEX label for FAM label on paper (for example) - Not all quenchers are dark (check compatibility) - Locked nucleic acids (LNA) bases, Minor groove binder (MGB) quenchers offer additional options → increase the affinity of the probe, which can result in shorter probes; may increase specificity (though not all the time) Bioinformatic tools for assay design and analysis: → input the sequence which you want your primers to anneal and specify the desired amplification conditions - Primer3Plus (Design): list of potential assays that may work given the criteria you entered - PrimerBlast (Design and Specificity): to try and ensure that your designed primers do not amplify nonspecific regions - BLAST (Specificity) - Mfold (2* folding): particularly important when working with SYBR green; can ensure that secondary folding will not inhibit the annealing of the oligos What is the best way to choose sequences for primers and probes? a. Highlight some sequence and hope for the best b. Use a primer design program such as Primer3Plus c. Pick some from a published paper d. Ask the postdoc in your lab Answer: B - Keep in mind that not all published papers require thorough validation, so you can't always trust what is published. Whether you are using a design program or selecting one from a published paper, it's recommended to ensure they are accurate before using them with your experimental samples. Chapter 5: Validated Assays Wet lab experiments to prove: efficiency and specificity - Validation is a critical part of ensuring that the qPCR experiment is accurately measuring what you think is measuring → Order pre-validated assays - PreAmplification assays help when samples are limiting; expands the pool of cDNA so more data can be reported Validated assays: Robust > efficiency: 95-105% > R2>0.99 Easy to use > temperature optimized Specific > intron spanning > next generation sequence - for specificity What parameters should be tested for a validated assay? a. Amplification efficiency b. Cq value less than 30 c. Sequence specificity d. None of the above Answer: A and C - Amplification efficiency and sequence specificity are the most important parameters when validating an assay. It would be great for Cq values to be less than 30 for the best precision, but this is not always possible. A Cq value in the range of 30 to 35 may be fine if you have validated your assay up to that point. Course 4: Assay Optimization for RT-qPCR Chapter 1: Why we Optimize and Validate Optimization - determining the ideal PCR conditions for an assay to maximize sensitivity and specificity Validation - empirically justify the use of an assay to quantify a target nucleic acid Assay sensitivity: - Ensure that the relative differences between different samples are accurate - Accurately detect a target even if it’s at a lower level - To establish the lowest detection level at which the PCR target can be accurately quantified → the lowest concentration that can be distinguished from the background noise Assay specificity: - Ensures that the assay is quantifying only what is intended MIQE Guidelines: - Bustin et al. (2009) - Minimum Information for Publication of Quantitative Real-Time PCR Experiments - Gold standard for qPCR best practice Chapter 2: Getting Started with Optimization Optimization and Validation Tools - Temperature gradient: to find the optimal annealing/extension temperature of the production ; to find a temperature warm enough to give a specific product, but cool enough to not encumber the PCR amplification - Melt Curve/Gel: allows to ensure the reaction is amplifying a specific product - Standard curve: to establish the PCR efficiency and dynamic range of the assay Temperature Gradient - Optimize Annealing Temperature (Ta) - Oligonucleotide sequence → GC content may cause an increase in Tm, longer amplicons have higher Tm than shorter amplicons  - Oligonucleotide concentration - Cations present in the buffer (salt concentration) → may affect the attraction between the cDNA strands → monovalent (Na+) → Divalent (Mg2+) - Run the temperature gradient protocol and then examine the Cq values generated for the samples at different temperatures - Pick the warmest temperature that doesn’t increase the Cq value (optimal temperature) → if the temperature is too warm, the primers won’t bind efficiently and it will take more PCR cycles to amplify the product → warmer temperature reduces the chances of primer-dimers and any off target products Considerations for Optimization - Choice of reagents - Primer/probe concentration → adding primers at concentrations that are too high can sometimes lead to primer-dimer formation, whereas a concentration that is too low can reduce the efficiency of the PRC - Simplex vs multiplex → single target per well (simplex) is ideal for users who have an ample amount of RNA or cDNA product; otherwise, multiplex can be used for limited products I run a thermal gradient PCR for a new assay using a control sample. Based on the temperatures and Cq values below, which temperature is most likely to be optimal? a. 58 degrees, Cq = 24 b. 59 degrees, Cq = 24 c. 60 degrees, Cq = 25 d. 61 degrees, Cq = 26 e. 62 degrees, Cq = 27 Answer: B - It is important to select the warmest annealing temperature that does not increase the Cq value. The advantage of using a warmer temperature is going to reduce the chances of primer-dimers and any off target products. If you run the annealing temperature too warm, it will lower the efficiency at which the primers bind. This will lower the total PCR efficiency and will confound the results. Chapter 3: Getting Started with Validation PCR primer specificity - Extra bands in PCR gel would mean that the PCR was yielding unintended products and needed to be re-optimized or redesigned - Melt curve analysis can check for PCR specificity within the same PCR reaction volume → decline in fluorescence is due to changes in pH as buffer is heated → sharp drop in fluorescence shown at around 84C is caused by the denaturation of the PCR product, and the SYBR Green is coming unbound → SYBR green only binds to dsDNA and that it only fluoresces when it is bound → multiple drops indicates the presence of multiple products - Melt peak shows a positive peak which corresponds to a single product → multiple peaks may show multiple products, though this usually stems from issues in primer design → What to do: Redesign the assay Look for a pre-validated assay Try a warmer annealing temperature (goal is to have the warmest annealing temp.) → run at the end of qPCR SYBR reactions You run a qPCR SYBR green reaction with a melt curve on a new assay, and you notice multiple melt peaks. What should you do next? a. Move forward and run the qPCR assay with your experimental samples b. Redesign the assay and test the new replacement primer set c. Raise the annealing/extension temperature to try to eliminate the off-target product  d. Purchase a pre-validated qPCR assay for the intended target Answer: B, C and D - There are three possible correct answers here. People often try to raise the annealing/extension temperature to eliminate the off-target product. Sometimes this works, often it does not. When this does not work, the issue is one of primer design. If this occurs, it’s recommended to either redesign the assay and test the new primer set, or purchase a pre-validated qPCR assay and run it under those validated conditions. It is not recommended to continue the experiment unless you have done one of these three. Chapter 4: Validation Continued Standard Curve Analysis - Determine the dynamic range for the assay → thereby knowing the upper and lower limits of quantification - Determine the precision of the assay - Determine the efficiency of the amplification Dilution Series Setup - Serial dilution is typically 10-fold - This would be diluted into an appropriate matrix to replicate that of the unknown sample, meaning that any inhibitors present in your sample that may affect the dynamic range of the assay will be accounted for in the dilution experiment → even spacing in amplification curves: demonstrates the premise of quantification in real-time PCR → Cq value of the dilution against the log of the starting quantity: inverse linear relationship - As Cq value increases by 1, the starting quantity of target is reduced by half E: 90% < E < 110% → with 100% efficiency, product is perfectly doubling with each PCR cycle R2: >0.98 → low value suggests that the data does not have a linear relationship with the log of the starting quantity; unreliable experimental data with the assay Slope: -3.32 y-int: 40 What range of efficiency is generally considered ideal for a qPCR assay? a. >0.98 b. Anything over 100% c. 70% to 130% d. 110% - primer dimer, check lowest quantity on curve → choosing the lowest quantity on the curve R2 measures the linearity of relationship between two variables. Target R2 > 0.98 I run a standard curve and get an efficiency of 140%. Where should I look to troubleshoot the problem? a. Nowhere! 140% efficiency is amazing! b. The melt curve c. The GC content of the product d. Make sure the primer Tms are as close as possible e. The most diluted sample on my standard curve Answer: B and C - The Melt Curve: If you are running a CYBR Green reaction and have multiple products or an off-target product such as a primer-dimer, CYBR Green will not know the difference. It just binds to double-stranded DNA and could result in the appearance that your product is doing more than double. The most diluted sample on my standard curve: If you look at your standard curve, sometimes the final concentration (the most diluted sample), will fall off that best fit line. This will pull the slope down, resulting in a slope that is less negative. A less negative slope will make it look like the assay is more efficient than it actually is. If you have 140% efficiency, look at the slope of the line, standard curve, and melt curve. If you have an efficiency of 140%: - Check on the lowest quantity on the melt curve (as it has more sensitivity) - Efficiency is high as it includes other non-target products Chapter 6: Considerations for Optimizing Multiplex Assays Considerations for Multiplexing: - The cost vs benefits of multiplexing → probes are more expensive than SYBR green; more upfront work may be required to validate a multiplex than simplex - Appropriate reagents → multiplex reagents tend to have a greater concentration of resources (polymerase, dNTPs, etc.) and the PCR reaction is rendered at a higher volume - Validation of assays in multiplex → ensures that one assay is not out-competing another for resources, or that different assays are inhibiting each other through primer-dimer formation Benefits of multiplexing: - You have limited samples - To distinguish similar sequences within the genes - Minimize cost - Time efficient Multiplex optimization - ensure that the multiplex assays do not interact negatively with each other Protocol: separate assays, multiplex and NTC will be run at the optimized PCR conditions; examine amplification curves for each of them → if the Cq values differ significantly in simplex and multiplex wells, this may suggest that the assays won’t perform well together → after, run a standard curve with positive control template Which of the following lab personnel should consider probe multiplexing? a. Lab scientist working with very small amounts of RNA in her sample b. A postdoc wanting to save time by pipetting less c. A grad student who is hoping to impress his advisory committee d. A lab manager wanting to save money on reagents e. A postdoc wanting to quantify different mRNA splice variants Answer: A and E - Because of the complexity that goes into multiplexing, most scientists are doing so out of necessity. A lab scientist working with very small amounts of RNA would be a good case for multiplexing. The other possibility is if you are working with similar homologous sequences. You would need an additional oligo like probe to help distinguish between them. Probe multiplexing is not going to save you money because of the expense of running probes compared to SYBR Green and primer sets. It’s not going to necessarily save you a lot of time due to validation and optimization time needs.