20.109(S20):Perform qPCR experiment and continue RNA-seq data analysis (Day7)

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20.109(S20): Laboratory Fundamentals of Biological Engineering

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Introduction

To eliminate clutter, the basepairs between the DNA strands were omitted. An animation of this process is linked here.
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 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 proportionately to the increase in double-stranded product. In contrast, the data for traditional PCR are simply observed as a band on a gel.

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 CT values from your qPCR assay. The CT 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 CT. 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

Before reading through the protocol below, please review the video provided by Sigma-Aldrich which describes qPCR reaction and how this method is used to determine the amount of a specific transcript, or gene of interest, in a sample (linked here).

Part 1: Review qPCR experimental procedures

In a previous laboratory session you purified RNA from your DLD-1 cell culture and used the RNA to generate cDNA. Today you will use quantitative PCR to probe the transcription levels of your gene of interest.

Part 1a: Clean cDNA

  1. Retrieve your cDNA samples from the front laboratory bench.
  2. Add 100 μL of Buffer PB to each cDNA preparation.
  3. Place four QIAquick columns into collection tubes and carefully label each according to your cDNA samples.
  4. Transfer the entire volume of cDNA + Buffer PB (~120 μL) from each tube into the appropriate QIAquick column.
  5. Centrifuge at maximum speed for 30 s, then discard the flow-through.
  6. Add 750 μL of Buffer PE to the QIAquick columns, centrifuge at maximum speed for 30 s, then discard the flow-through.
  7. Centrifuge the QIAquick columns at maximum speed for 1 min.
    • This will remove any residual liquid from the columns.
  8. Move each QIAquick column into a clean, labeled 1.5 mL centrifuge tube.
    • Be sure to remove the caps so they do not break off in the centrifuge.
  9. To elute the DNA, add 30 μL of dH2O pH = 8 to the center of the column and leave at your benchtop for 1 min.
  10. Centrifuge at maximum speed for 1 min.
    • The liquid in the microcentrifuge tube is your cleaned cDNA.
  11. Alert the teaching faculty when you have finished and place your samples on the front bench. The teaching faculty will measure the concentration of your cDNA samples with the NanoDrop.

Part 1b: Prepare primers

While you were away the sequences for the gRNA you designed were submitted to Integrated DNA Technologies (IDT). IDT synthesized the DNA oligos, or primers, then lyophilized (dried) them to a powder. The following steps were used to resuspend your oligos. Please be sure to write down relevant primer information and calculations in your lab notebook.

  1. Write down the original amount of DNA that arrived from IDT (written on white board)
  2. Calculate the amount of nuclease-free water that was needed to give a stock concentration of 100 μM.
  3. Calculate the volume of each stock that was required to prepare a 100 μL of solution that contains each qPCR oligo at a concentration of 10 μM.
  4. Tubes were centrifuged containing your lyophilized qPCR oligos for 1 min.
  5. Primer stocks were resuspended in the appropriate volume of sterile water, vortexed, and centrifuged.
  6. Primer mix was prepared by mixing appropriate volumes of forward primer, reverse primer and water into one tube.

Part 1c: Prepare cDNA for quantitative PCR assay

In addition to probing transcript levels for a gene of interest, you will also use primers specific for TBP (TATA binding protein). TBP is general transcription factor, meaning that this protein is able to bind specific promoters to activate transcription of downstream genes. Specifically, TBP binds to the TATA box that is located upstream of several genes in eukaryotes. Because TBP expression is not altered by etoposide treatment we will use its transcript levels to normalize the gene of interest expression levels in the analysis.

  1. Label 4 eppendorf tubes according to the designations below:
    • A = DLD-1, TBP primers
    • B = DLD-1 +etoposide, TBP primers
    • E = DLD-1, gene of interest primers
    • F = DLD-1 +etoposide, gene of interest primers
  1. Using the concentration measurements from Part 2b, calculate the volume of each sample that contains 20 ng of cDNA.
    • If the volume is greater than 8 μL alert the teaching faculty.
  2. Prepare master mixes of your samples such that:
    • Each reaction should contain 20 ng of cDNA, 2 μL of the appropriate diluted primer solution, and water for a total volume of 10 μL.
    • The volume of water is calculated as 8 μL minus the volume calculated in Step #2 for each sample.
    • For each sample/primer set combination, prepare enough master mix for 3.5 reactions.
  3. Give the tubes with your master mixes to the teaching faculty

Each master mix will be used to prepare three reactions (10 μL each), such that your samples are run in triplicate. This will allow you to complete statistics as described below in Part #3. To each 10 μL of sample the teaching faculty will add 10 μL of 2X SYBR Green reagent.

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

Stage Cycles Details
1 1 95°C for 3 min
2 40 95°C for 15 sec
60°C for 30 sec
3 1 Melt Curve Analysis

Part 1d: Examine qPCR results

Before you can apply statistical tools to your data, you must first normalize the expression levels of your gene of interest. To account for any unintended biases in RNA purification and / or cDNA preparation, it is important to normalize the expression of the transcript of interest to expression of a housekeeping or constitutive gene. Ideally, the gene to which the data of interest are normalized is not responsive to the treatment tested. In our experiment, we used TBP as it is not responsive to etoposide treatment. How might you confirm this assumption?

  1. Review the data posted on the Class data page.
    • Remember you will be assessing DLD-1 and DLD-1 +etoposide samples for each gene target you selected on M2D6.
    • All samples were probed using primers designed to your genes of interest and TBP primers.
    • Each reaction was completed in triplicate. Note: these are technical replicates.
    • The data are represented as the 'threshold cycle' CT or amplification cycle at which SYBR Green fluorescent signal was detected.
  2. Normalize each gene of interest expression to TBP expression (ΔCT).
    • Subtract the TBP CT value from the gene of interest CT values using the appropriate treatment conditions.
  3. Exponentially transform each normalized value to the ΔCT expression.
    • ΔCT expression = 2-ΔCT.
  4. Below you will review how to best represent these data using statistical analysis.

Part 2: Practice statistical analysis methods

Statistics are mathematical tools used to analyze, interpret, and organize data. The specific tools that you will use are confidence intervals (CI) and the Student's t-test. To begin, review the following definitions:

  • Mean (or average) is defined as:
Sp17 20.109 M2D9 mean equation.png
  • With infinite data, the mean (χι) approaches the true mean (μ).
  • Standard deviation measures the variation in the data and is defined as:
Sp17 20.109 M2D9 stddev equation.png
  • With infinite data, the standard deviation (s) approaches the true standard deviation (σ).

Because standard deviation is only justified when sufficient data have been collected to generate a normal curve, you will use confidence intervals to report the likelihood that your results predict the true mean. A confidence interval is a defined interval that is calculated to define the true mean to a specified level of confidence. Simply, it is possible to define a range in your data set that likely contains the true mean based on the calculated mean.

  • Confidence interval is defined as:
Sp17 20.109 M2D9 CI equation.png

In your data, you should use the CI to generate error bars due the low n. Be sure to report which confidence level was used to calculate the intervals reported. So, what does this all mean in regard to the data you will report? As an example, if the calculated χι of a data set equals 80 au there is a 95% chance the μ is between 50 au and 110 au, where au = arbitrary units. And how does this relate to s? If you know the μ, the σ represents a 68% confidence interval.

Lastly, you will use Student's t test to report if your data are statistically different between treatments.

  • Student's t test is defined as:
Sp17 20.109 M2D9 tcalc equation.png

The value you calculate with the Student's t test equation is referred to as tcalculated. This tcalculated value is compared to the ttabulated value in the the t table (linked here), according to the appropriate n - 1 using the p-value for the two-tailed distribution (which assumes that you do not know how the data will shift). If the tcalculated value is greater than the ttabulated, then the data sets are significantly different at the specific p-value. So, what does this all mean in regard to the data you will report? As an example, if the tcalculated for a data set with n - 1 = 10 is calculated to be 3, then the data sets are different with a p-value ≤ 0.05 (given that the ttabulated is 2.228). Which means that there is less that a 5% chance that the data sets are the same.

  1. Using the information above you will graph the average expression value for each gene of interest, then calculate the 95% CI and p-value using the Student's t-test.
  2. With this information, graph your data with error bars and include information concerning any statistical significance.

Reagents list

  • QIAquick PCR purification kit (Qiagen)
  • qPCR primers:
    • TBP forward, 5' - CCACTCACAGACTCTCACAAC - 3'
    • TBP reverse, 5' - CTGCGGTACAATCCCAGAACT - 3'
    • sequences for gene of interest primers can be found here
  • 2X SYBR Green (BioRad)

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