Real Time PCR

The polymerase chain reaction (PCR) is a technique to amplify a single or a few copies of a piece of DNA generating thousands to millions of copies of a particular DNA sequence. This method relies on thermal cycling consisting of cycles of repeated heating and cooling of the reaction for DNA melting and en!ymatic replication of the DNA. The PCR machine was in"ented #y $ary %ullis in &'(). Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase reaction #uffer and four types of nucleotides (dATP dTTP d*TP and dCTP) are +ey components to ena#le selecti"e and repeated amplification. As PCR progresses the DNA generated is itself used as a template for replication setting in motion a chain reaction in which the DNA template is e,ponentially amplified. -n this method amplified product can #e "isuali!ed using gel electrophoresis at the end of amplification. .nli+e normal PCR real time PCR is the method to monitor the amplification reaction while PCR is running. -n real time PCR also called quantitati"e PCR (qPCR) the amount of amplified product is lin+ed to fluorescence intensity using a fluorescent reporter molecule. The point at which the fluorescent signal is measured in order to calculate the initial template quantity can either #e at the end of the reaction or while the amplification is still progressing (real/time qPCR). Real/time quantitati"e polymerase chain reaction (qPCR) differs from regular PCR #y including in the reaction fluorescent reporter molecules that increase proportionally with the increase of DNA amplification in thermocycler. There are two types of fluorescent chemistries for this purpose0 dou#le strand DNA/#inding dyes and fluorescently la#eled sequence specific pro#e1primer. 234R *reen - dye and Taq%an hydrolysis pro#e are the common e,amples for these two respecti"ely. The 234R *reen - method doesn5t need fluorescently la#eled pro#e1primer and costs much less than the Taq%an method. This protocol is e,plaining the use of the 234R *reen - method. The +ey equipment for qPCR is a speciali!ed thermocycler with fluorescence detection modules which is used to monitor and record the fluorescence in real/time as amplification occurs. -n this protocol we are using real time PCR machine %,677)p from 2tratagene .2A. A qPCR for gene e,pression measurement in"ol"es different steps from RNA isolation re"erse transcription qPCR assay de"elopment qPCR e,periment to data analysis. These steps are descri#ed #elow in detail.

RNA isolation and First strand cDNA synthesis: RNA isolation can #e done using RNeasy %ini $it (8iagen *ermany) for real/time PCR analysis as qPCR requires quality RNA. The quality and quantity of RNA sample ha"e to chec+ #y agarose gel electrophoresis and ND/&777 spectrophotometer (Nanodrop technologies .2A). .se & 9g of total RNA for the first strand cDNA synthesis. :or cDNA synthesis the reaction can carry out using Affinity2cript qPCR cDNA synthesis +it (2tratagene .2A) in ;7 9l of reaction "olume. A typical reaction for re"erse transcription is listed #elow :irst strand master mi, (;<) >ligo dT (7.&=g1=l) Total RNA RNase/free water Affinity2cript RT en!yme mi, Then incu#ate at ?;7C for &) min Denature the en!yme at ')7C for ) min 2tore the cDNA product at /;77C for later use. .sually you can dilute &7/)7 folds and then use & =l each as template for one qPCR reaction. Primer Designing: Primer designing is the most challenging step of setting up a new qPCR e,periment. @owe"er the a"aila#ility of numerous primer and pro#e design software programs coupled with a set of easy to follow design rules ma+es the process relati"ely simple and relia#le. Primers for qPCR can #e designed using "arious softwares freely a"aila#le on internet. @ere we are using software Primer8uest a"aila#le on -DT site (-ntegrated DNA technologies http011eu.idtdna.com12citools1Applications1Primerquest1) with default parameters. :urther these primers ha"e to chec+ for their uniqueness using 4AA2T against NC4- data#ase. .niqueness of the primer/pairs can also #e chec+ed #y "isuali!ing the dissociation cur"e after real/time PCR reaction. &7.7 =l 6.7 =l &.7 =g &7.7 =l &.7 =l

%i, the a#o"e and incu#ate at ;)7C for ) min to allow primer annealing.

qPCR Reaction: The real/time reaction set up will #e0 ?7 ng of cDNA sample as a template forward and re"erse primers at ;77 n% concentration and 234R green reaction mi, (2tratagene .2A). A typical qPCR reaction tu#e using 234R *reen - chemistry has components in ;) =l of "olume. 234R *reen - mi, Reference dye :orward primer Re"erse primer cDNA &;.) =l 7.6B) =l 7.? =l 7.? =l &.7 =l

Nuclease/free water &7.B) =l Note that 234R green mi,ture has optimi!ed amount of DNA polymerase dNTP reaction #uffer. The reaction should carry out in 'C/well optical reaction plate (A,ygen) in %,677)p qPCR system (2tratagene .2A) as per manufacturer5s instructions. Ta+e all due caution to pre"ent contamination from genomic DNA and pre"ious PCR product #y using micropipette tips with #arrier. Dach reaction has to perform in triplicate for each #iological sample and three #iological replicates for each sample to monitor technical as well as sampling errors. To minimi!e "ariation in pipetting always prepare master mi,tures containing e"erything e,cept for template first and then dispense to indi"idual wells or tu#es. Add templates at the final step. 2eal co"ers firmly. Collect all liquid at the #ottom #y #rief centrifugation. To normali!e the "ariation among the samples any gene e,pression of which is not changing under different tissues growth conditions and at different de"elopmental stages should #e ta+en as reference gene for internal control. Data analysis: The ;/EECT method is easiest way to do relati"e quantification as it uses only CT "alues. After performing qPCR reaction the threshold CT "alues will o#tain for each sample with gene/ specific and normali!er/specific primers. The ECT "alues can #e calculated #y su#tracting CT "alue of normali!er from CT "alue of sample. :urther EECT "alues were o#tained #y su#tracting ECT "alue of cali#rator sample from test samples. The relati"e mRNA le"el in actual sample will further calculate #y formula0 Relati"e mRNA le"elF; /EECT. 2teps for calculation are as follows0

&) Normali!e the CT of *ene of -nterest (*>-) to that of reference gene (ref) for #oth the test sample and the cali#rator sample. This is ECT0 ECT(test) F CT(*>- test) G CT(ref test) ECT(cali#rator) F CT(*>- cali#rator) G CT(ref cali#rator) ;) Calculate the difference #etween the ECT of the test sample and the ECT of the cali#rator so called EECT0 EECT F ECT(test) G ECT(cali#rator) 6) Calculate the e,pression ratio0 D,pression ratio (folds) F ;GEECT @owe"er this method has an often untrue assumption that #oth *>- and reference gene ha"e the same amplification efficiency a condition that is rarely met in practice. Dissociation (Melting Curve) Analysis: All products generated during the PCR amplification reaction are melted at ')HC then annealed at ))HC and su#Iected to gradual increases in temperature. During the incremental temperature changes fluorescence data are collected until the reaction reaches ')HC. The result is a plot of raw fluorescence data units "ersus temperature. This "iew of the data may appear difficult to interpret at first #ut the rapid linear decrease in fluorescence #etween (;HC and (?HC where the maIor product melts is o#"ious. -n analy!ing the "arious com#inations of primer you can see one pea+. The distinct melting pea+s indicate multiple PCR products in this assay. A fully optimi!ed assay will contain only a single melt product. The Cts from these wells should not #e trusted as accurate and no meaningful quantification can #e #ased on these data. The presence of a single homogeneous melt pea+ for all sample reactions confirms specific amplification. The data from this reaction are relia#le and meaningful for analysis and interpretation. Conclusion: Real time PCR #ased on dou#le stranded DNA dye (234R green -) is a powerful tool for gene e,pression assay and it is relati"ely easy to de"elop. Careful e,perimental design and correct data analysis will pro"ide relia#le results. @owe"er 234R green chemistry is not suita#le for multiple, qPCR assays where amplification of more than one target is performed simultaneously in a single reaction tu#e.