Week 2: RT-qPCR

Introduction

There are different ways to study gene expression in cells and tissues, i.e., Northern blot, dot blot, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). For the Northern blot, RNA is first isolated, separated by formaldehyde agarose gel electrophoresis, and then transferred onto a membrane, followed by hybridization and detection to reveal the size and quantity of target mRNAs. In a dot-blot, samples of RNA are bound to a membrane and then hybridized with a gene-specific probe to determine the amount of target mRNA. In RT-qPCR, the reverse transcription (RT) is driven by RNA-dependent DNA polymerases, so-called reverse transcriptase (RTases), which synthesize single-stranded complementary DNA (cDNA) from an RNA template.¹⁴ These enzymes are made by both prokaryotic and eukaryotic organisms, as well as in retroviruses. They are important tools for molecular biologists in various applications, including gene expression analysis. After RT, quantitative polymerase chain reaction (qPCR) then amplifies the cDNA, and the product is used to estimate the RNA quantity in the original sample.

Traditional PCR requires post-PCR analysis for product detection, i.e., gel electrophoresis and image analysis, after completing a large number of PCR cycles. One pair of PCR primers should produce one specific PCR product shown as a single band in the gel. Real-time or quantitative PCR (qPCR) combines PCR amplification and product detection into one step without gel electrophoresis, and the result is quantitative because it measures the PCR product after each PCR cycle.¹⁵

SYBR Green-based and probe-based are two common types of fluorescent detection in qPCR. SYBR Green, a DNA intercalating dye, is excited and fluorescent when bound to double-stranded DNA (dsDNA), and the fluorescence increases ~1000X upon binding (Figure 4-3, left).^16,17,18 The dye binds non-specifically to dsDNA, mainly the PCR product in the reaction. It does not bind tightly to single-stranded primers or cDNA. If a dsDNA template is used for the qPCR, it is present at a low concentration compared to the PCR products. Nevertheless, non-target products are also measured because SYBR Green binds to all dsDNA.^17,18 The primers can form short dsDNAs called primer dimers. It would be hard to interpret the data if a reaction contains both non-target products and primer dimers.

For the detection of qPCR product, an amplification curve of fluorescence vs. cycle number (Figure 4-4) shows the product accumulation over the entire PCR run and three phases of PCR amplification, exponential, linear, and plateau. Each PCR reaction is characterized by the cycle number when the target amplification is first detected (threshold) or when the fluorescence intensity exceeds the background, called cycle threshold (Ct), crossing point (Cp), or quantitation cycle (Cq).¹⁶

The probe-based detection, i.e., TaqMan probe, significantly increases the specificity of qPCR, where a sequence-specific oligonucleotide modified with a 5′ fluorescent reporter dye (FAM, VIC, or TET) and a 3′ fluorescent quencher (TAMRA dye) is present in the reaction.^16,17 When the probe is intact, the quencher suppresses the reporter’s fluorescence. During annealing, the probe hybridizes with the target DNA in a region between two PCR primers. In the extension step, the 5′ → 3′ exonuclease activity of a specific Taq DNA polymerase degrades the hybridized probe to release quencher suppression and allow fluorescence to be detected (Figure 4-3, right).^17,18 Unlike the SYBR Green method, the fluorogenic probes eliminate nonspecific PCR products to be amplified and increase specificity. In addition, using different dyes for probe labeling allows simultaneous amplification and detection of multiple targets in the same PCR tube (multiplex qPCR)^17,18 significantly saving labor and cost, as well as obtaining more results for samples with limited source material. Additionally, placing reference genes or controls with the target in the same reaction tube increases precision and reproducibility. It eliminates tube-to-tube or well-to-well variations or the variations due to the sample positions on the outer edge of a PCR plate caused by evaporation of content during the qPCR process.¹⁷

To confirm a single PCR product amplified by a specific primer pair in qPCR, we can perform melt curve analysis immediately after PCR by measuring the decrease in fluorescence as temperature increases. The resulting melt curve obtained can 1) assess the dissociation characteristics of the PCR product (dsDNA) during heating, 2) determine the specificity of the qPCR assay, 3) indicate the melting temperature (Tm) of a target, and 4) identify nonspecific PCR amplification. Each PCR product (amplicon) should produce a single peak as its Tm after melt curve analysis. Different PCR products produce different peaks with different Tm’s (Figure 4-5).¹²

Theoretically, PCR amplifies DNA exponentially, doubling the number of PCR products with each cycle. Exponential, linear, and plateau are three phases of PCR progress (Figure 4-3). Among them, doubling PCR product per cycle only occurs during the exponential phase because during this phase, 1) all reagents are in excess for amplification without inhibition, 2) the low amount of product does not compete with the annealing capabilities of primers, 3) a sufficient quantity of DNA polymerase is still highly active, 4) there is little to no accumulation of pyrophosphates, and 5) there is little self-annealing of the accumulating PCR product.¹⁹ As amplification continues, the PCR eventually ceases exponential amplification of the template, and eventually, a “plateau effect” occurs (Figure 4-3). The leading cause of PCR plateau formation is due to primer depletion, not product accumulation or degradation of reagents.

In qPCR, the greater the starting quantity, the faster a significant increase in fluorescent signal or Ct appears, yielding a lower Ct/Cp/Cq (Figure 4-6).¹⁹ However, due to the plateau effect, samples with the same initial templates can obtain different quantities of PCR product measured at the plateau phase and, thus, different Cts. In contrast, reactions containing different amounts of the initial template may yield the same Ct at the plateau phase. Thus, unlike traditional PCR, only the Ct collected at the exponential phase in the qPCR can be used to extrapolate the initial template quantity, and are more representative than data collected in the later phase.

In RT-qPCR, the Ct value obtained from qPCR can be converted to a cDNA quantity, which is then extrapolated to the RNA quantity in the original sample. Nevertheless, this requires high PCR efficiencies to ensure experimental accuracy, and “exponential efficiency assessment” is required for all qPCR assays. For the assay, a serial dilution of the cDNA or total RNA is prepared, and each dilution is subjected to PCR amplification.²⁰ The Ct is plotted against log (cDNA/total RNA quantity) to construct a standard curve for the best-fit linear equation: y = mx + b (y = Ct, m = slope of the standard curve, x = log (cDNA or total RNA quantity), b = y intercept) (Figure 4-7). The equation is used to calculate the theoretical efficiency: E = 10^-1/slope– 1. The efficiency can also be expressed as the % amplification efficiency = (10 ^-1/slope– 1) × 100%. At most, PCR efficiency in the exponential phase is expected to double per cycle, corresponding to an amplification efficiency of 100%, and the slope is -3.32. A good reaction should have an efficiency of 90–100%, corresponding to a slope between -3.58 and -3.32. If the slope is below -3.58, the PCR exhibits low efficiency (< 90%).

y = Ct

m = slope of the standard curve

x = log (cDNA or total RNA quantity)

b = y intercept

The method enables comparison of amplification performance across genes, including reference genes, within the same experiment after preparing dilution series for each gene.²¹ The dilution series of each gene is amplified in real-time, and the resulting Ct values are used to construct a standard curve. Figure 4-8 shows that, if the two resulting straight lines from two genes, A and B, are parallel or not, suggesting their efficiencies are equivalent (left panel) or not (right panel). The Ct values of the A gene can also be subtracted from those of target B. The difference in Ct values is then plotted against the logarithm of the template amount (bottom panel). If the slope of the resulting straight line is < 0.1, amplification efficiencies of the two genes are comparable.

Errors in slopes are common. The causes include, but are not limited to, inhibitors, contamination, pipette imprecision or improper calibration, and dilution error, all of which can also account for efficiency greater than 100% since exponential efficiency should not exceed 100%. Therefore, efficiency assessments using equations should be conducted with caution.

Although RT-qPCR is routinely used in gene expression studies, multiple problems are associated with the methodology and analysis, including standardizing sample preparation/storage, reference gene choice, control sample selection, primer design, and data analysis. The reproducibility and reliability of the results depend on experimental design and interpretation. Comparing data normalized with different reference genes can show substantial differences in the final results.²² In addition to having a similar abundance in gene expression to the gene studied, reference gene expression should remain constant across treatment conditions in an experiment regardless of tissue type or treatment. One crucial task in gene expression analysis is selecting proper reference genes for accurate interpretation of the results. MIQE guidelines recommend using ≥ 2 reference genes for all studies seeking publication.²³

The collected Cts are not directly converted to quantities for gene expression study with RT-qPCR. Multiple variants and extra steps are involved in the calculation steps. Firstly, the data are normalized with the endogenous control or reference gene for the starting sample mass. The results of the testing sample are also compared to a calibrator, i.e., a control sample. Normalizing to a reference gene and a control sample is done earlier by subtracting Ct values and evaluating ΔCt and ΔΔCt values in the 2^-ΔΔCt method,²⁴ which assumes equal efficiency (E) among assays. Below are steps determining the expression of a gene of interest (GOI) in the test sample as the % gene expression of the control. The equation applies to AtRBCS1A and AtRBCS3B genes studied in this lab exercise. The 2^-ΔΔCt method does not require a standard curve to estimate the quantities of the original cDNA that are necessary to extrapolate RNA quantity in the original sample. It reduces cost, lowers labor, and has high throughput. However, constructing the standard curve is valuable for testing range and assessing PCR efficiency. Finally, it is necessary to validate the length of the PCR product by agarose gel electrophoresis before outsourcing DNA sequencing to confirm its identity.

In addition to not requiring post-PCR processing, the advantages of using RT-qPCR in studying gene expression include real-time detection, accuracy, rapid analysis, high sensitivity for mRNA transcription profiling, and the ability to detect low-level and difficult-to-detect mRNAs. It also has a relatively high throughput if proper equipment is provided.  However, it has limitations, such as the requirement for expensive equipment and reagents, sound experimental design, and an in-depth understanding of normalization techniques for accurate conclusions, due to its extremely high sensitivity.

One-step and two-step RT-qPCR are two common methods for studying gene expression.²⁵ In two-step RT-qPCR, reverse transcription and qPCR reactions are carried out in separate tubes (Figure 4-9, left). The first-strand cDNA synthesis reaction can be primed with random primers, oligo(dT) primers, or gene-specific primers.  To have an equal representation of all targets and avoid the 3′ bias of oligo(dT) primers, random primers or a mixture of oligo(dT) and random primers are often used. It is extremely sensitive and more efficient. The cDNA obtained from reverse transcription can be used to study the expression of multiple genes later.  However, it is more time-consuming and may require more optimization. In contrast, one-step RT-qPCR carries out both RT and qPCR in a single tube. Gene-specific primers are used because oligo(dT) or random primers may generate nonspecific cDNA, which can reduce the amplification of the target gene (Figure 4-9, right). One-step RT-qPCR is usually less sensitive and does not provide cDNA for future use.

For this week’s lab exercise, the RT reaction will be conducted with the purified total RNA obtained from last week’s lab, followed by two-step qPCR. The data received (Cts) will be analyzed by the 2^–ΔΔCt method to calculate the AtRBCS1A and AtRBCS3B mRNA expressions in leaves of dark-treated plants as % expression of the control without dark-treatment.

Procedure

A. Reverse Transcription – The 1^st strand cDNA Synthesis

Use filter tips for the entire experiment. 

1. Make a 20 µl aliquot of 20 ng/µl of your total RNA in RNase-free sterile reverse osmosis (RO) water. This is a dilution step.
2. Prepare 220 ng RNA (11 µl) and add 1 µl of Primer mix (1 µl blend of random hexamers: anchored oligo-dT [3:1]) and heat to 70°C for 5 min.
3. Set up two “No reverse transcriptions (RTs),” one for the dark treatment and the other for the untreated control. For no RTs, replace the 1 µl of Verso Enzyme Mix with RNase-free sterile RO water.
4. Cool on ice for 1 min before mixing with 7 µl of the reaction mix prepared by the instructor and 1 µl Verso Enzyme Mix.
   

   Diluted RNA (20ng/µl)	11 µl
   Primer mix	1 µl
   Reaction Mix (7µl)
   5X cDNA buffer	4 µl
   dNTP mix	2 µl
   R.T. enhancer	1 µl
   Verso Enzyme Mix	1 µl
   Total	20 µl

5. Put in a thermocycler at 42°C for 60 min (cDNA synthesis) and then 95°C for 2 min (inactivation).
6. Add 180 µl of sterile R.O. water into the reaction.

B. qPCR

Since each group only set up one reverse transcription reaction, each odd-numbered group (control) needs to pair up with one even-numbered group (dark-treatment) to share each other’s cDNA for setting up qPCRs.

• One of the groups needs to set up qPCRs for the “No RT” control and dark treatment.
• Actin and 18S rRNA (two reference genes) are used as internal controls, and AtRBCS1A and AtRBCS3B are the genes studied.

1. Obtain a strip of eight 0.2 ml PCR tubes without labeling. Ensure the reaction is set up from A to H (left to right) on the PCR strip, as shown in the diagram below.
2. Add 4 µl H₂O and then 10 µl of Master Mix into each tube.
3. Add 4 µl of cDNA from the control into each of the first four tubes (A–D). Discard tip for each pipetting.
4. Add 4 µl of cDNA from the dark treatment into each of the last four tubes (E–H).
5. Add 2 µl of each primer pair (refer to Table 4-1 in Module 4 Equipment and Materials) to each designated PCR tube shown below. (Change tips and make sure that all the contents get into the bottom of each PCR tube!)
6. The same setups are conducted for the no RT control and no RT dark treatment.

PCR Cycle:

• 95°C for 2.5 min, 40 cycles of 95°C for 20 sec, 50°C for 30sec, and 72°C for 30 sec

Data Analysis and Discussion

1. What is the RNA concentration in the reverse transcription tube after adding 180 µl of H₂O? Show your calculation.
2. Why did we heat the RNA sample and the primer mixture at 70°C for 5 minutes (step 2) before reverse transcription?
3. Based on the results you obtained and what you have learned about the melt curve, discuss whether the four sets of primers worked or not.
4. We used random primers and oligo dT primers in our reverse transcription reaction.
   1. If the primer set used for qPCR following the reverse transcription is located at the 5′ end of the gene, which primer would be better for the reserve transcription? Why?
   2. If the primer set used for qPCR is located at the 3′ end of the gene, which primer is better to use? Why?
5. Use the qPCR data obtained from the lab, calculate expressions of AtRBCS1A and AtRBCS3B in leaves of the dark treated Arabidopsis plants as % of expressions in the control using actin and 18S rRNA as the references. Show your step-by-step calculation. (Hint: The equation is shown in this week’s Introduction.)