Selective Control of Primer Usage in Multiplex One-step Reverse Transcription PCR A Journal Article Review
Polymerase chain reaction or PCR is one of the most promising techniques in Molecular Biology nowadays, and a variant of it is the multiplex RT-PCR or reverse transcription PCR which describes the amplification of the ribonucleic acid (RNA) of interest into its subsequent DNA counterpart. RNA amplification, like many other techniques that involve sub-cellular components, requires a set of complex rules and strict protocols. Hence, effective execution of the multiplex RT-PCR requires two steps, a process which ensures accuracy of results but carries with it the notion that prolonged extracellular experimental process may elevate the chances of contamination of the substrates. However, despite the impending probability of contamination, both scientists and researchers are taking the risk of using multiplex RT-PCR because it is a valuable tool in naming specific human and animal pathogens, used for the detection of diseases and may be used for gene expressions relative quantification.
In order to address the risk for contamination of the usage of multiplex RT-PCR, simplification of the process is required but such kind of step will involve the intensive optimization of the PCR techniques that will avoid undesired PCR primer extension during the reverse transcription step. The researchers of this paper tried to address this issue by designing a PCR method that will make use of PCR primers which are generally composed of thermolabile phosphotriester modification groups, particles which decrease the chances of PCR primer extension during the reverse transcription period and allows for better control of the primer extension process. Consequently, the results of this study suggest that the availability of manipulable primers, whose control is dependent on temperature gradients, provides a better way of facilitating improved one-step RT-PCR processes in both singleplex and multiplex types. In general, the application of the findings of this study marks subsequent advances in the quantification of mRNA expression levels as a function of reduced PCR bias. These events, when applied along with the other PCR techniques, pose a very promising method of reducing problems on PCR reaction specificity.
General Background
In vitro production of DNA from RNA copies or reverse transcription was deemed to be possible by Howard Temin in the 1960s where he emphasized that the availability of enzymes that can create a complementary copy of the RNA into single-stranded DNA is the most necessary step. Such kind of enzymes are termed to be reverse transcriptase because they can make a complementary DNA (cDNA) from single-stranded RNA of specific viruses which will then be used as template strand for the production of double-stranded DNA. The discovery of this phenomenon provides a way to obtain additional knowledge and eventually prevent and combat life-threatening viral particles like the human immunodeficiency virus (HIV) which is associated for AIDS, human T-cell lymphoma virus which is the causative agent of T-cell leukemia, and Rous sarcoma virus which is the connected with avian diseases. Hence, the discovery of in vitro reverse transcriptase is one of the most monumental events in the history of viral investigations.
Technological advances in the 21st century have brought about subsequent improvements in scientific procedures including the investigation of infective viral particles the introduction of using polymerase chain reaction is one of these technologies. Polymerase chain reaction or PCR is the method of creating many copies of DNA segments that are contain in a test tube by supplying it with appropriate enzymes at their corresponding temperature requirements. The efficiency of this process relies on the thermo-stability of the enzymes that will be used for the DNA amplification process. In this sense, the application of PCR techniques to reverse transcription or RT-PCR also bears with it subsequent limitations which are composed of low sensitivity (Lieberman, 2002) and complex multi-step processes. These problems are designed to be addressed in the study by using optimizing the PCR process which will entail the usage of thermolabile primers. The fulfillment of this step will provide a novel way administering RT-PCR with greater efficiency and less associated errors.
Introduction of the Research
As mentioned earlier, RT-PCR is an effective method that is widely used for viral pathogen detection and identification of genetic diseases. Furthermore, multiplex RT-PCR is an advancement of this technique because it enables the detection of multiple RNA segments in single reaction and allows for the detection of various mRNA expression levels through the usage of real-time detection. However, the likelihood to acquire erroneous data using this method is considerably high because of the long handling time of the substrates. It is inferred that this long handling time will result to the insertion of primer-mimicking particles which will eventually result in off-target copying. In this manner, the researchers have devised several hypotheses to address this issue by citing some scientific approaches.
The first among these hypotheses is the concept that the drawbacks of RT-PCR can be reduced through the usage of Hot Start DNA polymerases which will take its effect on the more stringent temperature of the polymerase chain reaction. Another is the usage of more heat-stable reverse transcriptase like the SuperScript III which tends to reduce the events of PCR primer binding to off target segments of the complementary DNA (cDNA). Unfortunately, these two options are not free of associated limitations because they permit the presence of both PCR primers and RT primers in the same reaction, another formidable source of error for the multiplex RT-PCR method. With this mind, the researchers settled with the more effective method of addressing the drawbacks which is by optimizing one-step RT PCR performance. This method can be achieved by utilizing physical barriers such as AmpliWax or PCR Gems which will tend to create a separation between the two types of primers in the single tube and thereby lessen the probability of errors and contamination. To further improve this method, the usage of thermolabile protecting groups consisting of phosphotriester linkages will be utilized. It is assumed that the usage of more heat-sensitive primer modifications will allow stability of the RT primer elongation and block attachment of unmodified RT primers to the said segment. Consequently, the goal of this paper is to propose a method that will seek to improve the RT-PCR techniques which is of great importance for the validation of microarray-based gene expression profile.
Experiments and Results
To start the experimental stage of the study, the researchers have determined the appropriate combination of PCR and RT primer that will be sued for the study. At the initial phase, unmodified oligo or random decamer for the RT step and thermolabile primers for the PCR step were used. For the PCR primer, three literature-defined targets were exposed to singleplex one-step RT-PCR in order to compare the performance of unmodified or natural PCR primers against primers that are modified at the 3 terminal penultimate position and are attached with thermolabile phosphotriester groups. Results showed that the modified PCR primers manifested better RT-PCR performance. RT step, on the other hand, were observed to function at its best when used with unmodified oligo or random decamer primers. Moreover, the combination of Precision PCR primer modifications and an unmodified oligo or random decamer RT primer was found to manifest the highest RT-PCR specificity and was therefore chosen to be used for the subsequent one-step RT-PCR experiments.
The aforementioned combination of unmodified and Precision modified primers were subjected to three targets which are composed of ABC transporter proteins for the human samples. These are specifically composed of ABCA5, ABCA6, and ABCA7 genes which were subjected to both singlepex and multiplex conditions. In a singleplex set-up, the correct amplicon was amplified but in a multiplex set-up, off-target amplifications were relatively high for the unmodified primers. Likewise, simultaneous amplification of the three targets affirmed the higher specificity of the Precision modified primers as compared to unmodified PCR primers. Next, the unmodified primers were tested for efficiency in response to temperature changes. This procedure is supported by the fact that reverse transcription tend to function better at elevated temperatures. Hence, the researchers tested if complementary DNA synthesis is really improved when substrates are exposed to high temperature of about 550C. The reverse transcriptase that was used on this part of the experiment was the SuperScript III RT (SSIII RT), an enzyme that is certified to possess high stability at elevated temperature. Single-step RT-PCR revealed that SS III RT performance with unmodified primers showed more specificity at 550C than at 420C while multiplex RT-PCR showed that simultaneous amplification of the three targets was poor even the procedure was executed at high temperature and this was supported by the appearance of a number of primer dimer formation. Hence, it can be said that the performance of unmodified primers was alleviated upon exposure to high temperature but only for the case of single-step procedure. These tests were duplicated but with the usage of different enzymes such as Taq and Hot Start DNA Polymerases in order to check if the same result will be achieved. The researchers eventually found out that modified primers established more specificity as compared to unmodified primers.
In this case, findings suggest that the Precision modified primer correspond to favorable changes in the specificity performance of multi-step RT-PCR methods. To further test this, the researchers prepared RNA standards from in vitro amplified human genes of ABCA5, ABCA6, and ABCA7 which were diluted from approximately 101 to 108. These standards were used as template strands for the detection of mRNA amplification levels in various human cell lines of RNA tissues of the trachea, brain and other vital body parts. Standard curve of the gene amplification levels were also created and it was discovered that the experimentally derived values for the singleplex and multiplex copies of the RT-PCR were almost similar. Subsequent experiments sought to quantify the gene expression levels of the three target human gene as well as the effect of unbalanced target abundance for one-step and multiplex RT-PCR performance, and to finally check for the quantity of targets that can simultaneously be amplified using modified primers. In general, results gathered form these experiments suggest that the usage of thermolabile protecting group for one-step reverse transcription polymerase chain reaction allows greater specificity and a general improvement in the performance of both singleplex and multiplex methods.
Conclusions
The researchers concluded that the application of this study can be seen on the detection of viral pathogens and disease diagnosis. Furthermore, they said that the utilization of the CleanAmp primers provides a promising way of improving the specificity of complementary DNA during the RT-PCR step. It was also mentioned that the findings of this study will be of great use for the determination of relative levels of gene expression in various tissue assays. Consequently, the reviewer agrees to the way by which the conclusions were formulated. The researchers were able to pinpoint the application of their study and suggest the target beneficiary of this kind of technologythe general public. In these times of public health problems, the application of the findings of this research will surely be an effective tool in addressing the surge of highly contagious and deadly viral particles.
Future Directions
As mentioned earlier, the reviewer believes that this research in particular and all the other scientific studies which tackle the same issues will be of great importance to public health and disease control. This kind of study will help scientists to better scrutinize viral particles which may lead to the formulation of medication or medical procedures that can lessen, if not totally eliminate, the various types of infectious viral particles. Hence, researchers and scientists must continue to carry out studies like this in order to address issues in public health and medicine.
In order to address the risk for contamination of the usage of multiplex RT-PCR, simplification of the process is required but such kind of step will involve the intensive optimization of the PCR techniques that will avoid undesired PCR primer extension during the reverse transcription step. The researchers of this paper tried to address this issue by designing a PCR method that will make use of PCR primers which are generally composed of thermolabile phosphotriester modification groups, particles which decrease the chances of PCR primer extension during the reverse transcription period and allows for better control of the primer extension process. Consequently, the results of this study suggest that the availability of manipulable primers, whose control is dependent on temperature gradients, provides a better way of facilitating improved one-step RT-PCR processes in both singleplex and multiplex types. In general, the application of the findings of this study marks subsequent advances in the quantification of mRNA expression levels as a function of reduced PCR bias. These events, when applied along with the other PCR techniques, pose a very promising method of reducing problems on PCR reaction specificity.
General Background
In vitro production of DNA from RNA copies or reverse transcription was deemed to be possible by Howard Temin in the 1960s where he emphasized that the availability of enzymes that can create a complementary copy of the RNA into single-stranded DNA is the most necessary step. Such kind of enzymes are termed to be reverse transcriptase because they can make a complementary DNA (cDNA) from single-stranded RNA of specific viruses which will then be used as template strand for the production of double-stranded DNA. The discovery of this phenomenon provides a way to obtain additional knowledge and eventually prevent and combat life-threatening viral particles like the human immunodeficiency virus (HIV) which is associated for AIDS, human T-cell lymphoma virus which is the causative agent of T-cell leukemia, and Rous sarcoma virus which is the connected with avian diseases. Hence, the discovery of in vitro reverse transcriptase is one of the most monumental events in the history of viral investigations.
Technological advances in the 21st century have brought about subsequent improvements in scientific procedures including the investigation of infective viral particles the introduction of using polymerase chain reaction is one of these technologies. Polymerase chain reaction or PCR is the method of creating many copies of DNA segments that are contain in a test tube by supplying it with appropriate enzymes at their corresponding temperature requirements. The efficiency of this process relies on the thermo-stability of the enzymes that will be used for the DNA amplification process. In this sense, the application of PCR techniques to reverse transcription or RT-PCR also bears with it subsequent limitations which are composed of low sensitivity (Lieberman, 2002) and complex multi-step processes. These problems are designed to be addressed in the study by using optimizing the PCR process which will entail the usage of thermolabile primers. The fulfillment of this step will provide a novel way administering RT-PCR with greater efficiency and less associated errors.
Introduction of the Research
As mentioned earlier, RT-PCR is an effective method that is widely used for viral pathogen detection and identification of genetic diseases. Furthermore, multiplex RT-PCR is an advancement of this technique because it enables the detection of multiple RNA segments in single reaction and allows for the detection of various mRNA expression levels through the usage of real-time detection. However, the likelihood to acquire erroneous data using this method is considerably high because of the long handling time of the substrates. It is inferred that this long handling time will result to the insertion of primer-mimicking particles which will eventually result in off-target copying. In this manner, the researchers have devised several hypotheses to address this issue by citing some scientific approaches.
The first among these hypotheses is the concept that the drawbacks of RT-PCR can be reduced through the usage of Hot Start DNA polymerases which will take its effect on the more stringent temperature of the polymerase chain reaction. Another is the usage of more heat-stable reverse transcriptase like the SuperScript III which tends to reduce the events of PCR primer binding to off target segments of the complementary DNA (cDNA). Unfortunately, these two options are not free of associated limitations because they permit the presence of both PCR primers and RT primers in the same reaction, another formidable source of error for the multiplex RT-PCR method. With this mind, the researchers settled with the more effective method of addressing the drawbacks which is by optimizing one-step RT PCR performance. This method can be achieved by utilizing physical barriers such as AmpliWax or PCR Gems which will tend to create a separation between the two types of primers in the single tube and thereby lessen the probability of errors and contamination. To further improve this method, the usage of thermolabile protecting groups consisting of phosphotriester linkages will be utilized. It is assumed that the usage of more heat-sensitive primer modifications will allow stability of the RT primer elongation and block attachment of unmodified RT primers to the said segment. Consequently, the goal of this paper is to propose a method that will seek to improve the RT-PCR techniques which is of great importance for the validation of microarray-based gene expression profile.
Experiments and Results
To start the experimental stage of the study, the researchers have determined the appropriate combination of PCR and RT primer that will be sued for the study. At the initial phase, unmodified oligo or random decamer for the RT step and thermolabile primers for the PCR step were used. For the PCR primer, three literature-defined targets were exposed to singleplex one-step RT-PCR in order to compare the performance of unmodified or natural PCR primers against primers that are modified at the 3 terminal penultimate position and are attached with thermolabile phosphotriester groups. Results showed that the modified PCR primers manifested better RT-PCR performance. RT step, on the other hand, were observed to function at its best when used with unmodified oligo or random decamer primers. Moreover, the combination of Precision PCR primer modifications and an unmodified oligo or random decamer RT primer was found to manifest the highest RT-PCR specificity and was therefore chosen to be used for the subsequent one-step RT-PCR experiments.
The aforementioned combination of unmodified and Precision modified primers were subjected to three targets which are composed of ABC transporter proteins for the human samples. These are specifically composed of ABCA5, ABCA6, and ABCA7 genes which were subjected to both singlepex and multiplex conditions. In a singleplex set-up, the correct amplicon was amplified but in a multiplex set-up, off-target amplifications were relatively high for the unmodified primers. Likewise, simultaneous amplification of the three targets affirmed the higher specificity of the Precision modified primers as compared to unmodified PCR primers. Next, the unmodified primers were tested for efficiency in response to temperature changes. This procedure is supported by the fact that reverse transcription tend to function better at elevated temperatures. Hence, the researchers tested if complementary DNA synthesis is really improved when substrates are exposed to high temperature of about 550C. The reverse transcriptase that was used on this part of the experiment was the SuperScript III RT (SSIII RT), an enzyme that is certified to possess high stability at elevated temperature. Single-step RT-PCR revealed that SS III RT performance with unmodified primers showed more specificity at 550C than at 420C while multiplex RT-PCR showed that simultaneous amplification of the three targets was poor even the procedure was executed at high temperature and this was supported by the appearance of a number of primer dimer formation. Hence, it can be said that the performance of unmodified primers was alleviated upon exposure to high temperature but only for the case of single-step procedure. These tests were duplicated but with the usage of different enzymes such as Taq and Hot Start DNA Polymerases in order to check if the same result will be achieved. The researchers eventually found out that modified primers established more specificity as compared to unmodified primers.
In this case, findings suggest that the Precision modified primer correspond to favorable changes in the specificity performance of multi-step RT-PCR methods. To further test this, the researchers prepared RNA standards from in vitro amplified human genes of ABCA5, ABCA6, and ABCA7 which were diluted from approximately 101 to 108. These standards were used as template strands for the detection of mRNA amplification levels in various human cell lines of RNA tissues of the trachea, brain and other vital body parts. Standard curve of the gene amplification levels were also created and it was discovered that the experimentally derived values for the singleplex and multiplex copies of the RT-PCR were almost similar. Subsequent experiments sought to quantify the gene expression levels of the three target human gene as well as the effect of unbalanced target abundance for one-step and multiplex RT-PCR performance, and to finally check for the quantity of targets that can simultaneously be amplified using modified primers. In general, results gathered form these experiments suggest that the usage of thermolabile protecting group for one-step reverse transcription polymerase chain reaction allows greater specificity and a general improvement in the performance of both singleplex and multiplex methods.
Conclusions
The researchers concluded that the application of this study can be seen on the detection of viral pathogens and disease diagnosis. Furthermore, they said that the utilization of the CleanAmp primers provides a promising way of improving the specificity of complementary DNA during the RT-PCR step. It was also mentioned that the findings of this study will be of great use for the determination of relative levels of gene expression in various tissue assays. Consequently, the reviewer agrees to the way by which the conclusions were formulated. The researchers were able to pinpoint the application of their study and suggest the target beneficiary of this kind of technologythe general public. In these times of public health problems, the application of the findings of this research will surely be an effective tool in addressing the surge of highly contagious and deadly viral particles.
Future Directions
As mentioned earlier, the reviewer believes that this research in particular and all the other scientific studies which tackle the same issues will be of great importance to public health and disease control. This kind of study will help scientists to better scrutinize viral particles which may lead to the formulation of medication or medical procedures that can lessen, if not totally eliminate, the various types of infectious viral particles. Hence, researchers and scientists must continue to carry out studies like this in order to address issues in public health and medicine.