20.109(S17):Complete cell survival assay and examine transcript levels in response to DNA damage (Day 4)

Introduction

Today you will be completing two experiments for module 2. The cell viability assay will provide information concerning the conditional lethality of compound 401 (NHEJ inhibitor) and olaparib (PARP inhibitor). In addition, you will use quantitative PCR to assess the levels of specific transcripts in response to DNA damage.

Cell viability assay
The CellTIter-Glo Luminescent Cell Viability Assay is a method for quantifying the number of viable cells based on measuring the amount of ATP present. ATP is a proxy for the presence of metabolically active (alive) cells. In this assay, the cells are lysed and ATP is released from the active cells. In a reaction catalyzed by a propriety luciferase enzyme, luciferin, ATP, and oxygen result in oxyluciferin, AMP, PP_i, carbon dioxide, and light. The light product is then measured using a luminometer.

Quantitative PCR
Quantitative polymerase chain reaction (qPCR) allows researchers to monitor the results of PCR as amplification is occurring (this technique is also referred to as real-time reverse transcription-polymerase chain reaction or real-time PCR). During qPCR data are collected throughout the amplification process using a fluorescent dye. The fluorescent dye is highly specific for double-stranded DNA and when bound to DNA molecules the fluorescence intensity increases proportionate to the increase in double-stranded product. In contrast, the data for traditional PCR are simply observed as a band on a gel.

To eliminate clutter, the basepairs between the DNA strands were omitted. An animation of this process is linked here.

As depicted in the image to the right, the fluorescent dye binds to double-stranded DNA during the cycles of PCR. At the annealing temperature the primer (blue arrow) binds to the template (black line). During an incubation at the extension temperature the new copy of DNA (orange dashed arrow) is sythesized by the polymerase enzyme. The inactive fluorescent dye molecules present in the reaction (grey stars) bind to the newly generated double-stranded DNA and become activated (green stars).

These qPCR amplification curve data were generated by Sp14 20.109ers for another experimental module!

These qPCR melt curve data were collected by Sp14 20.109ers for another experimental module!

To assess gene transcript levels, you will examine the C_T values from your qPCR assay. The C_T values are displayed as an amplification curve following qPCR (these values are also given numerically). The initial cycles measure very little fluorescence due to low amounts of double-stranded DNA and are used to establish the inherent background fluorescence. As double-stranded product is produced, fluorescence is measured and the curve appears linear. This linear portion of the curve represents the exponential phase of PCR. Throughout the exponential phase, the curve should be smooth. Sharp points may be due to errors in reaction preparation or failures in the machine used to measure fluorescence. As mentioned previously, the first cycle in which the fluorescence measurement is above background is the C_T. During the later cycles the curve shows minimal increases in fluorescence due the depletion of reagents.

Following the qPCR amplification measurements, a melt curve is completed. Melt curves assess the dissociation of double-stranded DNA while the sample is heated. As the temperature is increased, double-stranded DNA ‘melts’ as the strands dissociate. As discussed above, the fluorescent dye used in qPCR associates with double-stranded DNA and fluorescence measurements will decrease as the temperature increases. In qPCR, the melt curve is used to confirm that a single amplification product was generated during the reaction. If additional products were present, the melt curve would presumably show additional peaks. Why might this be true? Can you think of a scenario where two different products would produce a single peak in a melt curve?

Protocols

Part 1: Complete cell viability assay

Part 2: Design quantitative PCR primers

Part 3: Setup quantitative PCR assay

1. Obtain XX PCR tubes from the front laboratory bench and label according to the sample code.
   • A=
2. Retrieve your cDNA samples from the front lab bench.
3. Calculate the volume of each sample needed for 10 ng of cDNA.
4. Prepare your samples such that they

1. • Each reaction should contain 10 ng of cDNA, 2 μL of your diluted primer solution, and water for a total volume of 10 μL. For each sample/primer set combination, prepare enough for 2.5 reactions.
2. Add your reactions to the plate on the front bench according to the map. You will add 12.5 μL to each well.
3. Once everyone has added their reactions, the teaching faculty will add 10 μL of the 2X Syber Green master mix to each well.

For your reference, the PCR cycling conditions are listed below:

Stage	Cycles	Details
1	1	95°C for 10 min
2	40	95°C for 15 sec 60°C for 30 sec 72°C for 30 sec
3	1	4°C hold

Reagents

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