Quantitation of mrna Using Real-Time Reverse Transcription PCR (RT-PCR)

Quantitation of mrna Using Real-Time Reverse Transcription PCR (RT-PCR)

Quantitative Real-Time RT-PCR Versus RT-PCR In Real-Time RT- PCR, DNA amplification monitored at each cycle but RT-PCR measures the amount of accumulated PCR product at the end of the program, Real-Time PCR is quantitative, traditional PCR is qualitative.

Real-Time PCR Instrumentation

Growing Use of Real-Time PCR for the Years of 1990 2004 http://advan.physiology.org/content/29/3/151/f1.expansion.html

PCR Steps (Overview) Sample (tissue/cell) Preparation, RNA/DNA Extraction/Purification, cdna synthesis, RT-PCR, Electrophoresis, Documentation, Data Analysis.

PCR (Overview) primers polymerase denat. anneal extend extend denat. anneal DNA etc 95 o C 55-60 o C 72 o C 95 o C 55-60 o C 72 o C Adopted from: http://www.dnalc.org/ddnalc/resources/pcr.html

What are the reagents in Real- Time PCR tubes DEPC (Diethylpyrocarbonate) water, Master Mix (Buffer, dntp, Mgcl2),???? Primers (Forward, Reverse), Amplicon (target sequence, sample), Termocycler (Real-Time PCR machine). Point: buffers should stay out of light- Why???.

Master Mix (qpcr Product Detection) There are many fluorescence detection strategies to monitor PCR product accumulation during PCR Those which h use a dye e.g. SYBR Green, Eva Green, Disadvantages: not specific detects all double-stranded products. Advantages: inexpensive, i convenient, just as sensitive as a fluorescent probe.

Master Mix (qpcr Product Detection) Those which use a sequence specific probe which is exactly complementary to the PCR product sequence. Many probe formats available ( Scorpion, FRET, M. beacon). Disadvantages: Expensive, need new probe for every gene measured, cannot do melting analysis to confirm PCR product identity. Advantages: Sequence specific detection, potential for multiplex qpcr.

By far the most common methods are SYBR Green detection, and hydrolysis probes assays (e.g. TaqMan).

Detection strategies SYBR Green Exciting light SYBR Green Fluorescent emission signal Fd Fd 3' 3' 5' Target strand kate kirwan 2003

Detection strategies SYBR Green http://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/pierce/real

Real-Time PCR Assay Design Incubate at 95 C, 5 min, Incubate at 95 C,1 min, Incubate at optimized annealing temp (for each primer set) 15S - 1min, Plate Read, Goto line 2 for 40 more times, Melting Curve from 50C to 95 C, read, every 1.0 C, hold 10S END.

Quantitative Real-Time PCR Amplification Plot What does these curves mean??

Cycle Threshold (CT Value) Threshold Lines Point: Placement of threshold line affects Cq (CT Value)

Cycle Threshold (CT Value) High Concentration ti Medium Concentration Low Concentration Point: RT-PCR is semi-quantitative

SYBR Green Melt Analysis Raw data plot: Fluorescence (F) drops with Temperature (T) increase as DNA melts and SYBR is released F Derivative data plot: Plots the change in F divided by the change in T. Peak occurs at maximum dissociation rate df/dt T Melting Point T Point: Melting peak temperature reflects product length and sequence (% GC).

Real-Time PCR- The Problem Annealing @57 o C NTC Pineal cdna Example of Primer Dimers Eliminate by increasing annealing temperature Annealing @59 o C NTC Pineal cdna

Real-Time PCR- The Problem

Question? How could we confirm that our Real-Time product is the one that t we are looking for?

Run on Gel If your melt curve looks like this, you know that there is more than one product marke r 710 488 403 Unintended product 325 241 Correct size product 189 157/ 146 109 66 BLAST search did not detect this ~350 bp template But what about this is this definitely one product?

How to Calculate Copy Number in Standards Example A PCR product with 83 bp long has concentration of 56.4 ng/ul: What is the MW of the PCR product? Average molecular weight of one bp = 660 Da MW of the PCR product = 83 bp 660 54,780 Da How many molecules of product are there in 1 ng? Avogadro s constant = 6.02 x 10 23 = molecules/mole and. 1 mole = MW in grams So 54,780g of the PCR product = 6.02 x 10 23 molecules As 1 ng = 10-9 grams 1ng of the product = 6.02 x 10 23 / 54,780 10-9 = 1.099 x 10 10 copies How many copies of the PCR product are there per µl? There were 54.9 ng in each µl and 1.099 x 10 10 copies/ng 54.9 1.099 x 10 10 = 6.3 x 10 11 copies/µl of purified PCR product. http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.par.

Quantification To be useful as a measure of quantity, Cq of a sample must be related to either: The Cq (or equivalent) of another sample (relative quantification) or The Cq of a set of known copy number The Cq of a set of known copy number standards (absolute quantification)

qpcr Quantification Methods C T values Cq(CT) values absolute Pfaffl delta-delta C T standard curve to convert to quantity relative to highest/lowest expression level approximation method

Absolute Quantification Requires synthesis of standards from either a plasmid or cdna - amplify cdna/plasmid, purify PCR product, quantify amount of standard, Requires the use of 6-7 wells/tubes for standards, Standards d can be used as inter-assay calibrators, Gives an indication of copy number/unit volume or reaction, Does not preclude relative quantification if desired, Provides an estimate of assay efficiency from the standard curve.

qpcr Assays using standard curves (SYBR Green) Always run 10 8 10 1 standard curves for absolute quantification NTC????? Typical assay: sensitive - 10 copies efficient - 95.3% wide linear range - 10 8 to 10 1 copies with correlation coefficient 0.9996 Crossing poin nt (CT) 40 35 30 25 20 15 10 5 0 R 2 = 0.9996 efficiency = 95.3% 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 1.0E+07 1.0E+08 Log copy number

The graph of Cq vs. template concentration (copy number) is linear therefore sample copy number in unknown cdna can be determined by measuring Cq 35 30 point Crossing 25 20 15 10 5 0 100 1,000 10,000 100,000 Log concentration (copies)

Relative quantification (Pfaffl, DCq) Based on the expression of a target gene relative to the expression of one or more reference genes. Use mathematical models to determine relative amounts of amplicon, with (Pfaffl) or without correction for efficiency (delta-delta C T ). Does not require the inclusion of a standard curve.

Real-Time PCR is comparably a relative method so the amount of target DNA/RNA can be compared to the amount of RF/HK(Reference /Housekeeping) gene. (?????)

Reference Genes What Are They? A good reference gene should not vary in it expression level irrespective of experimental conditions, There is no perfect reference gene the expression levels of ALL genes vary, Single vs multiple reference genes if the expression levels of multiple reference genes are compared then the most stable genes can be identified, Should be selected based on cell specificities.

Why Normalize? Factors that can affect quantification of mrna expression: Amount of RNA loaded into reverse transcription (RT) reaction, Integrity of mrnas in samples, Efficiency of RT reaction, Presence of PCR inhibitors or enhancers in a reaction tube, Efficiency i of PCR: primers, non-specificity it etc, Cycler uniformity, Experimental conditions!

Continued The concept behind normalization is to measure one or more stable reference genes in the same sample i.e. they have been processed in the same way as the gene of interest (GOI), The expression of these reference genes is then used to correct for differences in quantification that are not due to the experimental conditions.

Calculating Efficiency The standard curve can be used to calculate assay efficiency (diluted samples -cdnas - can also be used) The difference in Cq value for a 10-fold dilution should be ~3.3 cycles when the efficiency is 100% Similarly, a cdna sample diluted two-fold should yield Cq values approximately 1 cycle apart (or half the number of copies at each dilution) The equation for calculating l efficiency i by this method is: E=10 (-1/slope) -1

Advantages of Real-Time RT-PCR over other C&M Biology Methods The detection of less than five copies (perhaps p only one copy in some cases) of a target sequence, Real-time platforms are relatively quick, with some affording high-throughput automation, Fi ll l ti PCR i f d i l d ti Finally, real-time PCR is performed in a closed reaction vessel that requires no post-pcr manipulations, thereby minimizing the chances for cross contamination in the laboratory.

Limitations of Real-Time PCR Methods Real-time PCR is susceptible to PCR inhibition by compounds present in certain biological samples, To circumvent this problem, alternative DNA p, polymerases (e.g., Tfl, Pwo, Tth, etc.) that are resistant to particular inhibitors can be used.

Limitations to Real-Time RT-PCR Methods. RNA itself is extremely labile compared with DNA, Therefore isolation must be carefully performed to ensure both the integrity of the RNA itself and the removal of contaminating nucleases, genomic DNA, and RT or PCR inhibitors.

Limitations of Real-Time PCR Methods Collection, storage, and transport can lead to a variable quality of RNA templates, Conversion of RNA to cdna is suggested to reduce its degradation. d

Limitations of Real-Time PCR Methods Improper assay development, incorrect data analysis, or unwarranted conclusions, Real-time PCR primer sets must be designed and validated by stringent criteria to ensure specificity and accuracy of the results, False positives or negatives must be considered when designing an assay to detect pathogens, Amplification and melting curves must be visually inspected while independent calculations based on these curves should be double-checked for accuracy.

qpcr tips and tricks TO AVOID CONTAMINATION: Decontaminate bench set-up area with 3% bleach Change gloves frequently ALWAYS have a separate area for setting up qpcr, well away from the area used for gel analysis purification of PCR products use a different room if possible!

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