Quantitative RT-PCR on Lighty cycler 480

From Wikionchus

Contents 1 Introduction to QPCR and the LC480 2 Setting up QPCR reactions 3 Interpretation and use of amplification and melting curves 4 Melting curves 5 Calculation of amplification efficiencies

Introduction to QPCR and the LC480

Quantitative PCR is a nowadays commonly used method to determine expression levels of transcripts. In brief cDNA is synthesized from complex RNA (either with random hexamer priming or gene specific primers) and a PCR reaction is performed which includes a sensor for amplification. The simplest (and in many people`s opinion best) method to measure amplification is based on the flourescent dye SybrGreen. SybrGreen binds in a completely sequence-unspecific manner to double stranded DNA. It is non-flourescent when not bound to DNA. A typical QPCR reaction is saturated with SybrGreen and therefore the increase in flourescence is a good measurement for the increase in double stranded DNA (i.e. amplification). We use the LC-480 SybrGreen PCR mix supplied by ROCHE, which contains everything needed for PCR except for (of course) cDNA and Primers. As SybrGreen binds to any double stranded DNA it is crucial to run the PCR with very stringent annealing temperatures (usually 60°C) and to include negative (without RT) controls for every sample. In addition, the melting curve, automatically generated by the LC480 cycler, is a very helpful tool for judging the quality of experimental data. I ll cover that in more detail in the last section of this protocol.

Setting up QPCR reactions

A single QPCR reaction consists of:

• 5 µl cDNA
• 10 µl LC480 PCR Mix
• 1 µl Primer F 10 mM
• 1 µl Primer R 10 mM
• 3 µl H₂O

Calculate how many samples you want to run in parallel and calculate for pipetting errors. You need roughly :

10 samples for the standard curve of the internal standard amplicon (beta-tubulin, 2 X 5 concentrations, usually run on sequential 1:10 dilutions of mixed stage cDNA) 2 x the number of your samples (run in duplicate) for both an internal standard (usually beta-tubulin) and your experimental amplicon. 1 x the number of your sampes for negative (-RT) controls.

• Load a PCR program-template from your personal account folder.
• Enter the sample designations into the LC480 software (GET AN INTRODUCTION by either Adrian, Arturo or Benjamin).
• Prepare the MasterMix for QPCRs and aliquot 15 µl each into the wells corresponding to the ones you entered into the software.
• Add 5µl of cDNA.
• Seal plate with special translucent and super-sticky foils, being careful not to touch the surface. Use the special tool to apply the foil.
• Spin down in plate-spinner. Be careful with the metal inlets of the centrifuge and make sure they are properly placed into the rotor.
• Run PCR !

Interpretation and use of amplification and melting curves

Amplification curves are a graphical represenatation of the efficiency of the amplification. The x-axis is cycle number (in principle it is a time plot of the extension times) and the y-axis is flourescence intensity. What the machine does is to measure the emitted flourescence of SybrGreen in the PCR tube with epiflourescent illumination in the second half of the extension time of each cycle, and to then calculate a continuous curve.

What you want:

• Steep curves - the steeper the curve the better the amplification efficiency (which can reach a theoretical maximum of 2). I have reached 1.94. But anything above 1.8 is considered "good".
• Curves that cross the crossing point (CP) after the 15th and before the 35th cycle.
• Curves that saturate !

What you do not want:

• Flat curves - flat curves mean bad amplification efficencies. (That makes trouble during relative quantification ! )
• Curves that cross the CP before the 15th cycle (too much template, high background due to the linear amplification of the original template) or after the 35th cycle (these are considered "BAD by default" by the program.
• Flatliners - amplicons that do not amplify. This occurs but should not - considering that the reagents for QPCR (not including RT stuff) cost ~ Euro 1 per sample.

Melting curves

The melting curve is measured at the end of the QPCR run. It is a separate program-module, that is inlcuded in all templates. SybrGreen flourescence is dependent on binding to dsDNA. Increasing the temperature leads to melting of the dsDNA strands - leading to decreased SybrGreen flourescence. The melting curve is a plot of flourescence (y axis) versus increasing temperature (x axis). This plot is not so easy and intuitive to interpret as the first derivative-plot. It shows the change of flourescence per temperature unit. Therefore peaks occur at those temperatures where the QPCR product melts. Perfectly amplified QPCR products show one (one and no more) peak in the melting curve. If primer dimers or non-specific products are amplified you can see that from multiple peaks in the melting curve. The melting curve per se does not show you how long the amplified product is. You should therfore run 2% gels on your amplicons and check whether they are the right size. However you can recognize primer-dimers in the melting curve because of their extremely low melting temperature peaks (around 55°C). As a comparison: a 100bp QPCR product melts at aroung 75-85 °C; largely depending on GC content.

Calculation of amplification efficiencies

It an idealized case the number of amplified DNA molecules in a PCR (if n is the initial value) is 2n after the 1st round and 2n² after the second round - 2n^k after k cycles. As mentioned before the theoretical maximal amplification efficiency is 2. This is rarely achieved in nature. Lets assume the amplification efficiency is 1.9. Then after the first round of amplification the amount of DNA is 1.9n - and 1.9n^k after k cycles. The amplification efficiency is simply speaking the "steepness" of the amplification curve. The LC480 uses a trick to calculate amplification efficiences: a standard curve of the internal standard is run and measured. This is usually a row of 5 serial dilutions of some standard template. For example the standard curve is made from cDNA at a concentration of 1, 10, 100, 1000 and 10000 (arbitrary units) - the machine then determines the Crossing Point (in the unit of amplification cycles); this is the saddle point of the amplification curve. It then plots the Crossing Points (CP, y axis) versus the concentrations of the standard curve reactions(x axis). The resulting points should define a line and the stepness of that line is a measurement for the amplification efficiency. For example: If two standards have a 10-fold difference in template concentration and the amplification efficiency is 2 then the one with lower concentration should need 3.32 cycles more to reach the CP.

(Thanks to Kyle Proffit for pointing out a mistake published in a previous version of this protocol@Wikionchus. 2^n = 10, n*log2 = log 10 log 10/ log 2 = n 1/ 0.301 = n n = 3.32.)