Fluorescence quantitative PCR is favored by more and more molecular biology researchers because of its convenient operation, fast operation and accurate experimental results. Today, when the experimental technology is mature, perhaps for you, Zui is not a problem in experimental technology - but which kind of PCR machine to choose - you need to consult the members of the laboratory, analyze the laboratory now and in the future The needs, balance the budget of the laboratory, and more detailed research on all kinds of tempting promotional materials. How do you choose to face different performance products?
First of all, I suggest that you get out of the two misunderstandings of fluorescence quantitative PCR:
Myth 1: The more instrument channels, the better.
With the maturity of PCR technology, multiplex amplification is becoming more and more lively, and the real-time PCR instrument is not immune. From the introduction of the single-channel real-time PCR instrument introduced by ABI at the beginning of Zui, all manufacturers have introduced 4-channel, 5-channel or even 6-channel fluorescence quantitative PCR instruments. The dazzling choice makes people feel at a loss. Someone accidentally walked into the channel. The more the better, the misunderstanding.
In the use of some manufacturers of 5-channel fluorescence quantifiers, in order to ensure the accuracy and accuracy of the experimental results, ROX (a fluorescent dye) or a dedicated Reference dye must be used in the experiment. These fluorescent dyes must be used alone. aisle. In this way, there are only four effective channels that can actually detect multiplex PCR fluorescence signals, and the use of calibration dyes may increase the cost of later use. Some of the detection channels of real-time PCR are only open for the proprietary fluorescent dyes or reagents of the manufacturer, and the effective detection channels are not as much as claimed in the publicity materials. It is especially important to confirm the effective detection channel of the real-time PCR instrument before purchase. You can't just listen to the publicity.
Considering the number of channels of multiplex PCR, it should also be based on the actual situation in the laboratory. Multiplex PCR is not suitable for everyone, and it is available to everyone because it complicates the experiment.
Myth 2: Real-time PCR machine does not require gradient function
For the quantitative PCR reaction using the dye method, although various PCR primer design software or empirical formulas are used to calculate the melting temperature (Tm value), the difference in the Tm value is large when the formulas used are different and the primer sequences are different. The melting temperature of the primer determines the annealing temperature. Moreover, the combination of bases in the template is ever-changing. For special fragments, the data obtained by the empirical formula may not be able to produce accurate results. The slight difference in annealing temperature may have a decisive influence on the results, so the "conditions" are once Very headache. The appearance of gradient PCR solves some problems. During the reaction, the temperature control conditions of each well can be changed according to the gradient within the specified range. According to the results, the suitable reaction conditions of Zui can be explored in one step.
Not only the annealing temperature, but also the denaturation temperature and the extension temperature can be optimized - this is very important for the amplification of many polymerase mixed enzymes such as Invitrogen, Clontech, Promega, most of the high-fidelity Taq enzymes, because the Taq and the correction enzyme are good. The reaction temperature may vary significantly, and optimizing the extension temperature is important.
Fluorescence quantitative PCR with gradient function can complete the optimization process that can be completed in many experiments in the past, which simplifies the cumbersome experiment of exploring the PCR reaction conditions, saves the experiment time and improves the efficiency, and saves the experiment cost.
When we go out of the wrong zone and want to choose a fluorescence quantitative PCR machine that suits our needs, what should we pay attention to?
1. Detection flux of the instrument When purchasing the quantitative PCR instrument, it is necessary to select different flux instruments according to the actual needs of the experiment. At present, the detection flux of real-time PCR on the market is as small as 16 to 384. If you do a general gene expression study or pathogen detection, you don't have to buy expensive instruments. The 96-well flux is sufficient; you need to look for instruments that require new drug targets and disease markers to consider purchasing 384-well plates. If you are unable to purchase a 384-well plate laboratory with limited funding, consider purchasing a 96-well plate fluorescence PCR instrument that runs fast (with the FAST module). With high-throughput instruments, you'll find sample preparation and small-system, multi-sample PCR systems built into flasks that limit experiment speed and results. Roche's MagnaPure accounting purification system can be used with Roche's Lightcycyler fluorescence quantitative PCR machine. Eppendorf's ep Motion5070, 5075 fully automatic workstation can solve the problem of nucleic acid purification, and can also be constructed in 96-well plate and 384-well plate PCR reaction system without adding the sample to the blackening of the eyes and hand cramps.
2, hardware design features Fluorescence quantitative PCR instrument mainly has a traditional 96-well plate type, innovative centrifugal type, etc., each design has its own unique but also has unavoidable shortcomings.
Fluorescence quantitative PCR of conventional 96-well plates can accommodate large sample sizes without the need for special consumables. Some traditional 96-well plate instruments use halogen-tungsten lamp excitation and CCD detection. These optical structures are on the top of a 96-well plate. Each sample hole has a different optical path from the source and detector—edge effects, resulting in results. influences. In order to ensure accurate and accurate experimental results, such instruments usually use ROX (a fluorescent dye) or a dedicated Reference dye as a reference calibration test result during the experiment. The short service life and high replacement cost of the tungsten halogen lamp have become a headache for many users. In the course of the experiment, the tungsten halogen lamp is used as a heat source, and more heat energy has a certain influence on the experimental results. The great advantage of CCDzui is that it can scan the fluorescent signal in all samples at the same time, but the sensitivity is low, and at the same time, there is interference in detecting the fluorescent signal between the samples. Fluorescent quantitative PCR produced by ABI and Bio-rad belongs to this category. However, Bio-rad's IQ series of real-time PCR has a gradient function.
Innovative centrifugal instruments usually use LED excitation and PMT detection, and the centrifugal design avoids edge effects. The LED light source is a cold light source that has no effect on the experiment, so there is no need to use other fluorescent dye calibration instruments, and the service life is long and does not need to be replaced frequently. The PMT can only collect a single fluorescent signal at a time, but the detection sensitivity is high. However, such instruments operate very fast, but they also have shortcomings such as small sample size, expensive consumables, no gradient function, and time integration. Corbett's Rotor-gene series and Roche's Lightcycler series are all such instruments.
With the use of real-time PCR technology, smart instrument manufacturers have made significant improvements in traditional 96-well plate instruments. First, Stratagene's Mx3000p broke through the use of CCD to detect fluorescence signals using PDT, which greatly improved the sensitivity of the experiment, but the excitation source still uses the traditional tungsten halogen lamp and has no gradient function. Eppendorf's Mastercycler ep realplex is a gradient-based real-time PCR instrument that uses 96 LEDs as the excitation source and uses advanced CPM (second-generation PMT) and 96-in-1 fiber as the detection system. The edge effect also ensures that the fluorescence signal between all samples does not interfere.
3, running speed In the many technical parameters of the fluorescence quantitative PCR instrument, the temperature rise and fall speed is also an indicator that manufacturers like to vigorously promote. Faster temperature rise and fall can shorten the reaction time, shorten the possible non-specific binding, reaction time, and improve the specificity of PCR. In order to complete the experiment better, various manufacturers have launched a real-time PCR instrument. For example, the Lightcycler 2.0 from ROCHE uses aerodynamics to complete 30-40 cycles in about half an hour. ABI's 7500 can be equipped with a FAST module with a 5°C/sec ramp temperature, which can be completed in 40-40 cycles in about 40 minutes. Zui near eppendorf's new Mastercycler ep realplex4S and 2S silver module fluorescence quantitative PCR instrument, the temperature of the temperature rise and fall can reach 6 ° C / sec and 4.5 / sec, is the current product in the same product temperature and temperature speed zui fast fluorescence quantitative PCR instrument . One would need 40-60 minutes of PCR on other instruments, eppendorf Mastercycler ep realplex4S and 2S (silver modules) only need to run for 28 minutes - don't underestimate the savings of tens of minutes, you can run more in one day Several rounds.
4, flexibility Large-scale busy laboratory equipment is enviable, but the regular laboratory can also have wonderful choices. Today's instrument suppliers have a large number of technical experts, and some can provide a basic research platform for molecular biology, not only to understand and meet your current needs, but also to take into account the needs you may change - upgrade and replace modules. Bio-rad's Mycycler can be upgraded to a quantitative PCR instrument. The ABI 7900 has the option of changing the 96-well slot to a 384-well slot. The centrifugal two-channel Rotor-gene3000A can be upgraded to a four-channel PCR machine for your research needs. Eppendorf is more thoughtful in this regard, such as the laboratory already has Mastercycler ep can be upgraded to the Mastercycler ep realplex PCR instrument according to your requirements, and there are four upgradeable models: 2-channel silver, aluminum module And 4-channel silver and aluminum modules. You can purchase and upgrade any Mastercycler ep realplex PCR instrument according to your own funding and experiment needs.
First of all, I suggest that you get out of the two misunderstandings of fluorescence quantitative PCR:
Myth 1: The more instrument channels, the better.
With the maturity of PCR technology, multiplex amplification is becoming more and more lively, and the real-time PCR instrument is not immune. From the introduction of the single-channel real-time PCR instrument introduced by ABI at the beginning of Zui, all manufacturers have introduced 4-channel, 5-channel or even 6-channel fluorescence quantitative PCR instruments. The dazzling choice makes people feel at a loss. Someone accidentally walked into the channel. The more the better, the misunderstanding.
In the use of some manufacturers of 5-channel fluorescence quantifiers, in order to ensure the accuracy and accuracy of the experimental results, ROX (a fluorescent dye) or a dedicated Reference dye must be used in the experiment. These fluorescent dyes must be used alone. aisle. In this way, there are only four effective channels that can actually detect multiplex PCR fluorescence signals, and the use of calibration dyes may increase the cost of later use. Some of the detection channels of real-time PCR are only open for the proprietary fluorescent dyes or reagents of the manufacturer, and the effective detection channels are not as much as claimed in the publicity materials. It is especially important to confirm the effective detection channel of the real-time PCR instrument before purchase. You can't just listen to the publicity.
Considering the number of channels of multiplex PCR, it should also be based on the actual situation in the laboratory. Multiplex PCR is not suitable for everyone, and it is available to everyone because it complicates the experiment.
Myth 2: Real-time PCR machine does not require gradient function
For the quantitative PCR reaction using the dye method, although various PCR primer design software or empirical formulas are used to calculate the melting temperature (Tm value), the difference in the Tm value is large when the formulas used are different and the primer sequences are different. The melting temperature of the primer determines the annealing temperature. Moreover, the combination of bases in the template is ever-changing. For special fragments, the data obtained by the empirical formula may not be able to produce accurate results. The slight difference in annealing temperature may have a decisive influence on the results, so the "conditions" are once Very headache. The appearance of gradient PCR solves some problems. During the reaction, the temperature control conditions of each well can be changed according to the gradient within the specified range. According to the results, the suitable reaction conditions of Zui can be explored in one step.
Not only the annealing temperature, but also the denaturation temperature and the extension temperature can be optimized - this is very important for the amplification of many polymerase mixed enzymes such as Invitrogen, Clontech, Promega, most of the high-fidelity Taq enzymes, because the Taq and the correction enzyme are good. The reaction temperature may vary significantly, and optimizing the extension temperature is important.
Fluorescence quantitative PCR with gradient function can complete the optimization process that can be completed in many experiments in the past, which simplifies the cumbersome experiment of exploring the PCR reaction conditions, saves the experiment time and improves the efficiency, and saves the experiment cost.
When we go out of the wrong zone and want to choose a fluorescence quantitative PCR machine that suits our needs, what should we pay attention to?
1. Detection flux of the instrument When purchasing the quantitative PCR instrument, it is necessary to select different flux instruments according to the actual needs of the experiment. At present, the detection flux of real-time PCR on the market is as small as 16 to 384. If you do a general gene expression study or pathogen detection, you don't have to buy expensive instruments. The 96-well flux is sufficient; you need to look for instruments that require new drug targets and disease markers to consider purchasing 384-well plates. If you are unable to purchase a 384-well plate laboratory with limited funding, consider purchasing a 96-well plate fluorescence PCR instrument that runs fast (with the FAST module). With high-throughput instruments, you'll find sample preparation and small-system, multi-sample PCR systems built into flasks that limit experiment speed and results. Roche's MagnaPure accounting purification system can be used with Roche's Lightcycyler fluorescence quantitative PCR machine. Eppendorf's ep Motion5070, 5075 fully automatic workstation can solve the problem of nucleic acid purification, and can also be constructed in 96-well plate and 384-well plate PCR reaction system without adding the sample to the blackening of the eyes and hand cramps.
2, hardware design features Fluorescence quantitative PCR instrument mainly has a traditional 96-well plate type, innovative centrifugal type, etc., each design has its own unique but also has unavoidable shortcomings.
Fluorescence quantitative PCR of conventional 96-well plates can accommodate large sample sizes without the need for special consumables. Some traditional 96-well plate instruments use halogen-tungsten lamp excitation and CCD detection. These optical structures are on the top of a 96-well plate. Each sample hole has a different optical path from the source and detector—edge effects, resulting in results. influences. In order to ensure accurate and accurate experimental results, such instruments usually use ROX (a fluorescent dye) or a dedicated Reference dye as a reference calibration test result during the experiment. The short service life and high replacement cost of the tungsten halogen lamp have become a headache for many users. In the course of the experiment, the tungsten halogen lamp is used as a heat source, and more heat energy has a certain influence on the experimental results. The great advantage of CCDzui is that it can scan the fluorescent signal in all samples at the same time, but the sensitivity is low, and at the same time, there is interference in detecting the fluorescent signal between the samples. Fluorescent quantitative PCR produced by ABI and Bio-rad belongs to this category. However, Bio-rad's IQ series of real-time PCR has a gradient function.
Innovative centrifugal instruments usually use LED excitation and PMT detection, and the centrifugal design avoids edge effects. The LED light source is a cold light source that has no effect on the experiment, so there is no need to use other fluorescent dye calibration instruments, and the service life is long and does not need to be replaced frequently. The PMT can only collect a single fluorescent signal at a time, but the detection sensitivity is high. However, such instruments operate very fast, but they also have shortcomings such as small sample size, expensive consumables, no gradient function, and time integration. Corbett's Rotor-gene series and Roche's Lightcycler series are all such instruments.
With the use of real-time PCR technology, smart instrument manufacturers have made significant improvements in traditional 96-well plate instruments. First, Stratagene's Mx3000p broke through the use of CCD to detect fluorescence signals using PDT, which greatly improved the sensitivity of the experiment, but the excitation source still uses the traditional tungsten halogen lamp and has no gradient function. Eppendorf's Mastercycler ep realplex is a gradient-based real-time PCR instrument that uses 96 LEDs as the excitation source and uses advanced CPM (second-generation PMT) and 96-in-1 fiber as the detection system. The edge effect also ensures that the fluorescence signal between all samples does not interfere.
3, running speed In the many technical parameters of the fluorescence quantitative PCR instrument, the temperature rise and fall speed is also an indicator that manufacturers like to vigorously promote. Faster temperature rise and fall can shorten the reaction time, shorten the possible non-specific binding, reaction time, and improve the specificity of PCR. In order to complete the experiment better, various manufacturers have launched a real-time PCR instrument. For example, the Lightcycler 2.0 from ROCHE uses aerodynamics to complete 30-40 cycles in about half an hour. ABI's 7500 can be equipped with a FAST module with a 5°C/sec ramp temperature, which can be completed in 40-40 cycles in about 40 minutes. Zui near eppendorf's new Mastercycler ep realplex4S and 2S silver module fluorescence quantitative PCR instrument, the temperature of the temperature rise and fall can reach 6 ° C / sec and 4.5 / sec, is the current product in the same product temperature and temperature speed zui fast fluorescence quantitative PCR instrument . One would need 40-60 minutes of PCR on other instruments, eppendorf Mastercycler ep realplex4S and 2S (silver modules) only need to run for 28 minutes - don't underestimate the savings of tens of minutes, you can run more in one day Several rounds.
4, flexibility Large-scale busy laboratory equipment is enviable, but the regular laboratory can also have wonderful choices. Today's instrument suppliers have a large number of technical experts, and some can provide a basic research platform for molecular biology, not only to understand and meet your current needs, but also to take into account the needs you may change - upgrade and replace modules. Bio-rad's Mycycler can be upgraded to a quantitative PCR instrument. The ABI 7900 has the option of changing the 96-well slot to a 384-well slot. The centrifugal two-channel Rotor-gene3000A can be upgraded to a four-channel PCR machine for your research needs. Eppendorf is more thoughtful in this regard, such as the laboratory already has Mastercycler ep can be upgraded to the Mastercycler ep realplex PCR instrument according to your requirements, and there are four upgradeable models: 2-channel silver, aluminum module And 4-channel silver and aluminum modules. You can purchase and upgrade any Mastercycler ep realplex PCR instrument according to your own funding and experiment needs.
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