Microfluidic PCR, qPCR, RT-PCR & qRT-PCR
1. Introduction about microfluidic PCR, qPCR, RT-PCR & qRT-PCR
From New England Biolabs website (1)
The most common DNA analysis are the Polymerase Chain Reaction (or PCR) and Quantitative Polymerase Chain Reaction (qPCR). Both methods consist in the specific exponential amplification of DNA sequence.
PCR and qPCR have numerous applications, some directly and others indirectly implied in other molecular techniques like DNA sequencing for example. One of the direct PCR applications is obviously the cloning of DNA sequences (e.g. gene).
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It also can help to make detectable low amounts of DNA (e.g. for pathogen detection, like bacteria or virus).
As for biological analysis in general, microfluidics present numerous advantages for PCR analysis:
- Whole biological process integrated and thus simplified for the end-users
- High-throughput, multiplexed and highly parallel assays
- Faster analyses due to the shorter reactions and/or separation times
- Portable devices for point-of-care applications
- Low reagent consumptions
- Global cost reduction per analysis
In this review, we will present the PCR, qPCR and other associated methods with their microfluidic applications. For complementary information, the excellent review from Zhang et al. (2) can also be read.
2. PCR and its applications in microfluidic devices
a. PCR Principle
PCR requires several reagents in a common mix:
- DNA sample: the original DNA from which a specific sequence will be amplified
- Primers: a pair of short nucleotides sequences (10-20 base pairs) corresponding to the partial sequences at the two extremities of the specific amplified DNA sequence. They will thus determine the specific amplified sequence.
- Nucleotides or more precisely deoxynucleotide triphosphates (dNTP): basically, the 4 types of DNA bases (A,T,C,G) which will be used to synthesize the new DNA amplicons (i.e. the new DNA samples resulting from the PCR process).
- DNA polymerase: an enzyme that enables to create the new DNA strand from single-stranded DNA as a template by assembling specifically the nucleotides.
Once the mix is prepared, the PCR basically consists in temperature cycles between 2 or 3 different temperatures for each PCR phase:
- Denaturation: this step is carried out at 94-98°C and causes DNA melting, i.e. the separation of the DNA strands yielding two separate single-stranded DNA.
- Annealing: this step is carried out at 50-65°C to allow the annealing of the primers to the single-stranded DNA templates at the specific locations corresponding to the two extremities of the specific amplified DNA sequence. Annealing phase can be made at the same temperature as the extension phase to make a PCR using only 2 different temperatures.
- Extension: this step is usually carried out around 70°C. The temperature mainly depends on the DNA polymerase used since it corresponds to the phase where the DNA polymerase binds to the primer/single-strained DNA complex to synthesize the new complementary DNA strand by incorporating the surrounding nucleotides.
b. PCR microfluidic devices and applications
It can be noted that PCR relies mainly on temperature cycles. The critical technique for integrated device is therefore their heating method that will be thoroughly discussed in the next paragraph.
We will focus here on general PCR microfluidic devices.
Among DNA amplification methods, PCR was the first one developed in microfluidic chip since it is the most common and simple nucleic acid amplification method. Indeed, when PCR is carried out on chip to take advantage of microfluidics benefits (speed(3,4), parallelization(4) and sensitivity(5)), its result analysis can be easily performed off-chip.
Then more integrated devices comprised also the subsequent DNA analysis, i.e. mostly electrophoresis (see review about microfluidics for DNA analysis) (6–8).
Multiple integrated PCR chambers, from Gong et al., Biomedical Microdevices, 2006 (4)
Microfluidic PCR chip with integrated electrophoresis analysis, from Pal et al., Lab on Chip, 2005 (7)
3. Thermalisation and temperature measurement systems in microfluidic devices
It can be seen from PCR process that temperature cycles and thus the thermalisation system is the essential technical part of PCR devices.
Also, even if temperatures can only be calibrated before experiments, it is preferable to monitor and control temperatures during PCR for a precise and stable sample thermalisation.
In this paragraph, we will therefore discuss the thermalisation and temperature measurements techniques implemented in microfluidic PCR devices.
a. PCR thermalisation in microfluidic devices
i. Thermoelectric techniques for PCR thermalisation in microfluidic devices
For integrated microfluidic devices, it is convenient to use thermoelectric techniques since they are readily compact. Indeed, many PCR devices and lab-on-chip used Peltier elements directly put in contact with their microfluidic devices to heat locally their PCR chamber (9–12). Peltier and other heating blocks present the disadvantage to have a relatively important thermal mass and slower temperature ramping rates (2). To overcome this problem, samples can be rapidly displaced between heating elements at fixed temperature, instead of constantly changing the temperature of heating elements. This process called flow-through PCR is explained in a specific paragraph.
To further miniaturize the PCR microfluidic devices and accelerate thermal process, thin films of metal (mostly Platinum) (7,13) or polysilicon (3) can be directly integrated in the microfluidic devices during its fabrication.
Thin film heaters in PCR chip, From Lee et al., Journal of Micromechanics and Microengineering, 2005 (13)
ii. Optical systems for PCR thermalisation in microfluidic devices
Optical systems present the advantage to not induce any thermal mass and can thus accelerate thermal transitions (2). Several systems use infrared (I.R.) lamps and their infrared radiations to heat PCR samples (6,14). Thus, rapid thermalisation has been carried out to obtain 15 PCR cycles in only 240s (14).
Compared to I.R. lamp, laser straightforwardly yield focused lights with higher intensities. This way it can be more easily used for miniaturized devices and nanoliter samples (15–17). This technology enabled to carry out up to 40 PCR cycles in 370s.
For the cooling phase, these optical systems often use air cooling through fan or only air contact in open system (this latter comprising so intrinsic problems for PCR contaminations).
It can also be noted that these optical systems require cumbersome setups that hinder their integration.
Laser heating of PCR droplets film, From Kim et al., Optics Express, 2009 (16)
iii. Thermalisation air and liquid flows for PCR thermalisation in microfluidic devices
To accelerate temperature transitions, instead of displacing the PCR samples like in flow-through PCR systems or changing the temperature of heating elements, heating flows can be displaced and alternated on a static PCR chamber.
The original PCR system developed by Wittwer et al. used this method using hot air cycling (18) which has been later applied to PCR microfluidic chips (19).
Compared to air, heating liquid flows can also be used for their improved thermal conduction that enables to reach higher thermalization speeds. This method has been applied in microfluidic devices (20,21).
With similar approach, using Elveflow ultra-fast pressure controller and fluorescence reader and based on the Elveflow ultra-fast temperature control (22), ELVESYS developed an ultra-fast qPCR microfluidic system for the molecular detection of diseases like Anthrax and Ebola in less than 8 minutes with a detection efficiency identical to commercial systems that are 7 to 15 times slower (23). The microfluidic device also uses the advantage of a large surface to volume ratio inherent to microfluidics to enhance thermal exchanges. In addition to ultra-fast and sensitive qPCR, the system uses monophasic samples and is compatible with a broad sample volume ranging from nanoliters to tens of microliter.
Elvesys system for ultra-fast microfluidic qPCR
b. Temperature measurement for PCR in microfluidic devices
i. Thermoelectric temperature measurements for PCR in microfluidic devices
Thin films are also widely used as temperature sensors in microfluidic device (2,22) once again for their integration properties. In PCR microfluidic devices, they can be easily integrated in parallel of thin films heaters (3,7,13). Same materials like Platinum and polysilicon can be used for the sensing parts. In the case of thin films, the measured temperature is therefore a surface one. Otherwise, larger structures like thermocouple can be used within the PCR chamber volume (9).
Nevertheless, in both cases, thermoelectric temperature sensors present the disadvantage to be directly in contact with PCR samples which can induce contamination and interaction with PCR samples. Also, most of the time, the thermoelectric temperature sensors only measure locally the PCR temperature. For this latter problem, larger thermoelectric structures like interdigitated ones can be used to measure average temperatures (3).
Interdigitated thermoelectric sensors for an average temperature measurement. The sensors are here integrated with the thermoelectric heaters. From Erill et al., Journal of Micromechanics and Microengineering, 2004 (3)
ii. Optical temperature measurements for PCR in microfluidic devices
Since thermoelectric structures enable rather local temperature measurements, it may be preferable to use optical techniques that can perform an average measure more easily.
Thermochromic Liquid crystals (TLC) has been used for PCR temperature characterization before microfluidic assays (24,25) but, to our knowledge, no systems used TLC during the actual PCR process for real-time temperature measurement. The probable interference of TLC with molecular reagents may be problematic.
For temperature characterization, infra-red pyrometry can also be used for non-contact and straightforward measurement, like it has been made for the ELVESYS ultra-fast qPCR system (23). Pyrometry has also been used for real time measurement during PCR assays even if, in this case, the absence of optical setup for real-time measurement of DNA amplification in parallel simplify the experimental setup (26). Nevertheless, pyrometry remains a surface measurement method unlike TLC.
Thermosensitive fluorophores can also be employed for PCR measurement like rhodamine B. Interestingly, they can yield local or global temperature measurement to map thermally microfluidic (27) and are compatible with real time temperature measurement during PCR assays (28).
TLC temperature measurement of a microfluidic device. From lles et al., Lab on Chip, 2005 (29)
4. qPCR and its applications in microfluidic devices
a. qPCR principle
Quantitative PCR (qPCR) is also called real-time PCR but can easily be confused with reverse-transcript PCR (RT-PCR, for RNA analysis).
qPCR relies on the same principle than PCR instead that the PCR product is detected in real-time during the amplification process thanks to fluorescent reporters associated with adapted optical excitation and detector.
These fluorescent reporters can either be fluorescent intercalating agents like Sybr Green or specific fluorescent probes.
Intercalating agents react with double-stranded DNA which makes this fluorescent DNA detection non-specific but cheaper. Therefore, this type of detection requires another step to verify the amplification specificity: a melting curve analysis. During this step, fluorescence is monitored while the temperature is increased. As the temperature is raised, DNA strands dissociate and thus the fluorescence, specific to double-stranded DNA, decreases. If the derivative of the fluorescence in function of temperature reveals a single-peak, the PCR reaction can be considered as specific. If several peaks appears, it means that non-specific PCR products have been unintentionally amplified (like primer dimers for example).
Melting curve analysis for qPCR with intercalating agent
Specific fluorescent probes are short nucleotide sequences, specific to the amplified DNA sequence, with fluorescent report at one end a quencher of fluorescence at the opposite end. The probes anneal to the single-stranded DNA at the same time than primers, during the annealing phase and are degraded when they are reached by the DNA polymerase during the elongation phase. This type of detection is therefore highly specific detection without any further step.
Fluorescent probes mechanism
Compared to PCR, qPCR presents the advantage to be faster (automated detection during PCR), more sensitive, have a much broader dynamic range and a better repeatability.
b. qPCR microfluidic devices and applications
Like for PCR, microfluidic devices enabled to accelerate thermalisation process for fast qPCR process (40 qPCR cycles in 20min and approximately 1h with commercial standard systems), helped with smaller PCR working volume, while conserving the same amplification efficiency and sensitivity (i.e. minimum nucleic acid concentration detectable) than regular commercial systems (30,31).
Microfluidic device was also developed for parallel pathogen detections (water parasites) using capillary injection and preloaded primers to simplify greatly system complexity and end-user task (32).
Microfluidic qPCR device. Adapted from Ramalingam et al., Sensors and Actuators B, 2010 (32)
5. RT-PCR & qRT-PCR and their applications in microfluidic devices
a. RT-PCR & qRT-PCR principle
PCR can be employed in DNA detection but it can also be used to detect RNA by simply adding a previous step to the PCR process. Indeed, RNA structure is very close to the DNA one but contain ribose instead of deoxyribose and an uracil base instead of thymine. Also, in most of its biological role, RNA is a single-stranded molecule and is much shorter than DNA sequences. Main applications of RNA quantification by RT-PCR is gene expression analysis from messenger RNA (mRNA) and virus detection (many viruses are RNA viruses).
In the 70s, reverse transcriptases, i.e. enzymes that enable the reverse transcription of RNA in DNA, were discovered. Thus, by using PCR biochemical reagents comprising a reverse transcript polymerase and a previous thermalisation step around 50°C, complementary DNA (cDNA) can be obtained from RNA, cDNA which can be then amplified like in a regular PCR process.
Differences between DNA & RNA
In the same way than PCR and qPCR, RT-PCR product can be detected in real-time during amplification for a qRT-PCR process. It is important here to distinguish RT-PCR and qPCR. RT-PCR could be previously used for both Reverse Transcription Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction. So using the RT-PCR could easily be confusing about the mentioned process. So now Quantitative Polymerase Chain Reaction and its abbreviation qPCR is rather used instead of Real-Time Polymerase Chain Reaction. A Quantitative Reverse Transcription Polymerase Chain Reaction is so abbreviated qRT-PCR.
RT-PCR can be performed either in a one-step assay combining Reverse Transcription and PCR in a single reaction chamber or two-steps separating the two processes in different reaction chambers. The main advantage of the one-step process is its simplicity reducing manipulation bias. On the other hand, one-step process requires to use only sequence-specific primers also compatible with the following PCR step. The main advantage of the two-steps process is the possibility to use different types of primers like Oligo(dT)s and Random primers. Briefly, the main advantage of Oligo(dT)s primers is the specific generation of full length cDNA from poly(A)-tailed mRNA from eukaryotic cells and the main advantage of random primers is its high cDNA yield from all types of RNA.
RT-PCR: different RT primers types. From Biology Department – Davidson College website. (33)
RT-PCR: one and two steps processes. From Life technologies website. (34)
b. RT-PCR & qRT-PCR microfluidic devices and applications
Two-step qRT-PCR can also be used to carry out the RT step off-chip of viruses and then the PCR with a microfluidic chip to accelerate the process duration (30 cycles in 15 min (31), 40 cycles in 17 min (35)) while conserving commercial system efficiency and sensitivity (31) or decreasing them (35).
One-step qRT-PCR has been made also in polymeric microfluidic chip associated with thermal and optical system but yielding a decreased efficiency compared to commercial systems (36). Other integrated system has carried out one-step qRT-PCR on chip with biphasic samples by decreasing the reaction time up to 35min for 50 cycles (37), cycle threshold was compared to commercial systems but not the efficiency neither the sensitivity.
Originally, RT-PCR product can also be detected within microfluidic chip by colorimetry using a immunochromatographic strip like used classically in pregnancy test (38). This type of system simplifies greatly the detection setup since it does not require any optical analysis.
Microfluidic RT-PCR assay associated with immunochromatographic (or lateral flow) detection. From Kim et al. Biosensors and Bioelectronics, 2012 (38).
6. Microfluidic Continuous Flow/Flow-through and Oscillating flow PCR
a. Microfluidic Continuous Flow/Flow-through and Oscillating flow PCR Principle
As mentioned in the paragraph concerning the thermoelectric techniques for thermalisation, to overcome the drawback of large thermal mass inherent to Peltier elements and heating blocks, instead of changing their temperature, PCR samples can rapidly be transferred to different heating blocks maintained at the PCR temperatures corresponding to the different phases. Thus, very high thermalisation speeds can be reached, especially if PCR mix volumes are also reduced.
Unlike in static systems, it is therefore preferable to use material with high thermal mass like plastic (e.g. Polycarbonate, PMMA, etc..) for these devices to avoid temperature variations, promote temperature uniformity and reduce power consumption (39).
Microfluidic Continuous Flow/Flow-through system for 30 PCR cycles with the different heating zones pointed out. From Moschou et al., Sensors & Actuators B, 2014 (39).
Nevertheless, this system present several drawbacks. Due to their specific design, it is complicated to easily adjust the different PCR phases times and the PCR mix volumes. Since the heaters temperatures are fixed, melting curves cannot be obtained. Lastly, it is also complicated to associate this technique with pre-PCR phases like cell lysis and DNA purification (see microfluidics for DNA analysis review). For that, the simpler method it to use the same mix for the cell lysis and PCR (40,41) even if it can deteriorate the amplification quality. Using more complex lab-on chip devices, cell lysis and DNA purification with solid phase extraction method has been carried out before continuous flow PCR (42).
b. Microfluidic Continuous Flow/Flow-through and Oscillating flow PCR devices and applications
Basically, two types of systems using displacing PCR mix have been conceived.
The first one consists in serpentine configuration with a number of PCR cycles fixed intrinsically by the design (39,41): continous flow or flow-through PCR. As previously mentioned these devices enable very fast PCR like 40 cycles in 120s (43).
Their main drawback of this technique is the inability of these devices to change the number of PCR cycles. Electrochemical detection associated with continuous flow PCR has been also used to detect DNA amplification instead of using electrophoresis or optical detection (40).
The second type consists of oscillating the PCR mix between the different temperature zones: oscillating flow PCR. Thus, any number of PCR cycles can be set for the amplification solving the drawback of the continuous-flow PCR systems.
This technique was first carried out in a capillary (44) but was then adapted in microfluidic chip using Quake valve to circularly circulate the PCR mix between the different temperature zones and even realize an integrated quantification of the DNA amplication (44). With this technique, very fast DNA amplification can be also obtained: 30 PCR cycles in 6 minutes (45).
Microfluidic Oscillating flow PCR chip. The 1st RTV layer correspond to the PDMS Quake valve and the second one to the PDMS microfluidic channels for PCR sample. From Chiou et al., Analytical Chemistry, 2001 (44).
7. Microfluidic Digital PCR
a. Microfluidic Digital PCR principle
With the development of microfluidics, emerged the field of digital microfluidics dealing with emulsion and droplets within microfluidic devices.
The precursor paper of Beer et al. (46) showed the interest of this technique to handle ultra-low amount of DNA within droplets and thus increase even more the detection limits obtained with qPCR. Indeed, with 10 pL droplets, a single DNA copy could be amplified. At the end of qPCR reaction, fluorescent droplets are counted and Poisson distribution is finally used to determine thus the absolute number of templates in droplets.
Now, digital PCR (dPCR) is commercialized through several companies and is specially used for ultra-sensitive detection. Nevertheless, dPCR requires longer thermalisation times to obtain a homogenous thermalisation between all the generated droplets after their transfer in PCR tube.
Precursor digital microfluidic device, from Beer et al., Analytical Chemistry, 2007 (46)
b. Microfluidic Digital PCR devices and applications
As previously mentioned, the interest of dPCR was first demonstrated to surpass conventional qPCR sensitivity with single DNA detection. The interest of this improved sensitivity was then also demonstrated for other analysis techniques requiring PCR steps like Copy Number Variation (CNV) detection which is involved in numerous diseases.
For example, dPCR was demonstrated more sensitive than qPCR to detect CNV in breast cancer related gene (HER2) (47). Using mutation-specific amplification with dPCR, rare mutated genes could also be detected in the presence of a 200 000-fold excess of unmutated genes (48).
Large number of droplets acting like independent micro-assays can be rapidly assessed (e.g. 1 millions (49)) for high-throughput PCR assays. Multiplexed PCR-based detection detection can be also carried out in droplets by varying the fluorescent dyes concentrations (48,50).
Digital PCR for rare mutation detection. From Pekin et al., Lab on a Chip, 2011 (48)
8. Microfluidic single cell PCR & RT-PCR
The advantage of single cell PCR/RT-PCR is the possibility to quantify and discriminate precisely the genetic expression of every single cells, even if they represent relatively a very small population among the global cell population, without losing it in the averaged genetic expression obtained by assessing all the cells at the cell type. Very rare biological events can be thus detected.
a. Single cell PCR & RT-PCR with “Continuous” microfluidic devices
In the previous paragraph, we saw the development of digital PCR using droplets to digitalize the PCR mix. We use here the term “continuous” to differentiate the microfluidic devices using no droplets to isolate single cells and molecules.
Quake’s group is a precursor in genetic analyses of single cells. Using the already mentioned Quake valves to isolate multiple reaction chambers and inject sequentially PCR reagents, the first integrated microfluidic for gene expression analysis (i.e. RT-PCR analysis) with off-chip electrophoresis analysis (51,52). The same method has been applied for stem cell expression analysis (53). Interestingly, by comparing microfluidic devices (using also Quake valve) and macroscopic methods for the analysis of RNA from single cell (10 pg), microfluidic devices showed a much better sensitivity to detect gene expression: 74% of genes with microfluidic devices vs 4% with regular method due to the dilution issues for the RT phase (54). Fluidigm commercializes a single cell RT-PCR system based on this technology.
Single cells has been also captured by hybridization on a functionalized microscopic surface at the cell size. Then, post-RT PCR analysis has been also carried out on-chip by capillary electrophoresis (55). Microfluidic devices can also be used for larger scale single cells genetic expression analysis (300 cells per run) and on-chip real time amplification detection (56). Instead of droplets, single cells can rapidly be captured in micro-cavities at large scale (hundreds of cells) by diluting properly the cell suspension and using an oil−surfactant system (like digital PCR) to isolate properly the PCR chambers and carry out then qRT-PCR from single cells (57).
Single cell RT PCR analysis, from White et al., PNAS, 2011 (56)
b. Single cell PCR & RT-PCR with Digital microfluidic devices
Since the precursor paper of He et al. showing the encapsulation of single cells in droplets and their subsequent lysis (58), it was pretty obvious the PCR analysis of single cells within droplets would be then soon doable.
One of the first works to carry out qRT-PCR from single cells in droplet was made by Mary et al. (59) analyzing E-cadherin gene expression. Another approach was developed using agarose droplets (60) facilitating their manipulation for post-amplification analysis which could be so easily carried out by flow cytometry or microscopic observation. Thus, the expression of a cancer biomarker gene, EpCAM, has been distinguished between different cancer cells.
Compared to the “continuous” microfluidic devices, the main advantage of digital/droplet microfluidic for single cell RT-PCR is the possibility of high-throughput analysis. As an example, Eastburn et al. developed microfluidic droplet system enabling the independent analyses of 50 000 cells in a single experiment (61).
Microfluidic droplet qRT-PCR devices for single cell analysis from Eastburn et al., Analytical Chemistry, 2013 (61).
9. Conclusion about microfluidic PCR, qPCR, RT-PCR & qRT-PCR
Microfluidics has been widely used for the different DNA amplification process (PCR, qPCR, RT-PCR). The microfluidic devices enable to accelerate PCR processes, reduce reagent consumption, reach high-throughput assays and integrate pre or post-PCR assays on-chip.
Commercial microfluidic DNA amplification systems start to appear even if they are often more expensive than regular systems and use different consumable types which can slow down their integration within the scientific communities. The recent most successful microfluidic application and transfer is the digital PCR.
The definitive transfer of microfluidic PCR technologies will require methods easily transferable to large scale production and so low-cost consumables associated with an easy end-user oriented workflow.
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For more tutorial about microfluidics, please visit our other tutorials here: «Microfluidics tutorials». The photos in this article come from the Elveflow® data bank, Wikipedia or elsewhere if precised. Article written by Timothée Houssin (June 2015).
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