Both RT-PCR and Qualitative PCR (Endpoint) are essentially the same PCR process. The difference comes that RT-PCR collects signal (fluorescent) information at the end of each amplification cycle building the amplification curve where with endpoint PCR you are limited with the signal at the end of the process. After RT-PCR is finished the product in the plate can be run on the gel as endpoint PCR amplicons.

Endpoint PCR, in general, can only reliably answer if this genetic sequence determined by the primer pair is present in the sample. RT-PCR, however, can detect the difference in abundance.

For example, you have the 3 samples of transgenic mice: with the gene of interest presented as follows: +/+, +/-, and -/- if you run endpoint PCR in most cases you get positive bands in first and second samples and no band in third. You will not be able to reliably determine between +/+ and +/-. If you run the RT-PCR however on the same samples you will clearly see the difference in the amplification curve.

I.e. if we use RT-PCR and add histone primers group as our housekeeper and do the normalization the values for the example sample would be as follows: +/+ (1.0) , +/ (0.5)-, -/- (0 no amplification). Of course, since it is relative abundance we can choose any sample (exempt 0) to be our 1 (or 100 if you like). For example, if we choose our +/- to be 1 the values change to +/+ (2.0) , +/- (1.0), -/- (0 no amplification) representing the same results.

In most common use, however, the RT-PCR utilized to measure gene expression through extracting the total RNA from the sample, conversion of RNA to c-DNA and then amplification of c-DNA with PCR chemistry with measurement of fluorescence at each step. If amplification curves are normalized to the housekeeping genes such as GAPDH or B-actin relative abundance can be determined as fold change value.

Some scientists on the limited budget even try to use quantification with the endpoint PCR in a similar way using 4 primers in the mix for the target and housekeeping genes and then performing relative quantification of the bands on the gel. (the same way Western Blot samples are quantified). It sometimes works, but it is not a very sensitive method or with RT-PCR being cheap and available not very well accepted by the scientific community. In the past, I have seen people doing it.

In theory, you can replicate the RT-PCR curve process using standard PCR and agarose gel (i.e. without RT-PCR machine). You will need to prepare at least 6 (More is more reliable) or so samples and then run 6 amplification programs for the progressive increase of the number of cycles i.e. stop the program after 5, 10, 15, 20, 25, 30. Then run all samples on the gel and see at which cycle you get the band visible on the gel this will be your Ct value (cycle threshold). And then run the same process for your housekeeper to make sure that housekeeping gene bands will show up in the same cycle if there is a difference in housekeeping band than you to make a correction for the cycle number i.e. if one sample produce band for housekeeper earlier than other than there is a difference in loading that need to be normalized. Naturally, this method is very resource and time/effort consuming and not very reliable. I believe back in the 1980es people might have tried to do something like this but nowdays everything is automated.

In the context of viral infections, the difference between the endpoint and RT-PCR is the following: Endpoint PCR will let you know if the virus present or absent even if there is a few copies of the virus after 30 cycles PCR will produce the signal, 2^29 is a huge amplification power if you have single virus sequence after 30 cycles you will get 536,870,912 copies.

If you run RT-PCR for the virus you can determine the viral load. The earlier cycle threshold will indicate more virus copies present in the initial sample and more severe infection. For example, you have 3 patients samples and one healthy control. You got Ct values: 8, 10, 30 and 0 (no signal) for control. These results will clearly indicate that patient 1 with (ct value 8 has the most severe infection) as the sample was amplified to be detectable only after 8 cycles amplification 2^7 meaning very high initial copy number, patient 2 with ct value 15 is has a mild infection and the last patient have very few copies of the virus that was amplified only after 30 cycles 2^29. If you run PCR products on the gel you might even see these differences if they are explicit. i.e. the first patient band would be brighter. (that is the meaning of qualitative PCR). However, RT-PCR allows you to distinguish very subtle changes. I don't think it is very meaningful to use any housekeeping gene when working with the virus material so you can operate directly with the ct values. There are few techniques where you can convert ct values to the viral copy numbers, this was determined in the literature for most known viruses usually by building the linear or log curve of the known copy numbers and projecting the ct values to the curve.

I hope it helps your understanding of the PCR and RT-PCR and its use with the viruses.

Both RT-PCR and Qualitative PCR (Endpoint) are essentially the same PCR process. The difference comes that RT-PCR collects signal (fluorescent) information at the end of each amplification cycle building the amplification curve where with endpoint PCR you are limited with the signal at the end of the process. After RT-PCR is finished the product in the plate can be run on the gel as endpoint PCR amplicons.

Endpoint PCR, in general, can only reliably answer if this genetic sequence determined by the primer pair is present in the sample. RT-PCR, however, can detect the difference in abundance.

For example, you have the 3 samples of transgenic mice: with the gene of interest presented as follows: +/+, +/-, and -/- if you run endpoint PCR in most cases you get positive bands in first and second samples and no band in third. You will not be able to reliably determine between +/+ and +/-. If you run the RT-PCR however on the same samples you will clearly see the difference in the amplification curve.

I.e. if we use RT-PCR and add histone primers group as our housekeeper and do the normalization the values for the example sample would be as follows: +/+ (1.0) , +/ (0.5)-, -/- (0 no amplification). Of course, since it is relative abundance we can choose any sample (exempt 0) to be our 1 (or 100 if you like). For example, if we choose our +/- to be 1 the values change to +/+ (2.0) , +/- (1.0), -/- (0 no amplification) representing the same results.

In most common use, however, the RT-PCR utilized to measure gene expression through extracting the total RNA from the sample, conversion of RNA to c-DNA and then amplification of c-DNA with PCR chemistry with measurement of fluorescence at each step. If amplification curves are normalized to the housekeeping genes such as GAPDH or B-actin relative abundance can be determined as fold change value.

Some scientists on the limited budget even try to use quantification with the endpoint PCR in a similar way using 4 primers in the mix for the target and housekeeping genes and then performing relative quantification of the bands on the gel. (the same way Western Blot samples are quantified). It sometimes works, but it is not a very sensitive method or with RT-PCR being cheap and available not very well accepted by the scientific community. In the past, I have seen people doing it.

In theory, you can replicate the RT-PCR curve process using standard PCR and agarose gel (i.e. without RT-PCR machine). You will need to prepare at least 6 (More is more reliable) or so samples and then run 6 amplification programs for the progressive increase of the number of cycles i.e. stop the program after 5, 10, 15, 20, 25, 30. Then run all samples on the gel and see at which cycle you get the band visible on the gel this will be your Ct value (cycle threshold). And then run the same process for your housekeeper to make sure that housekeeping gene bands will show up in the same cycle if there is a difference in housekeeping band than you to make a correction for the cycle number i.e. if one sample produce band for housekeeper earlier than other than there is a difference in loading that need to be normalized. Naturally, this method is very resource and time/effort consuming and not very reliable. I believe back in the 1980es people might have tried to do something like this but nowdays everything is automated.

In the context of viral infections, the difference between the endpoint and RT-PCR is the following: Endpoint PCR will let you know if the virus present or absent even if there is a few copies of the virus after 30 cycles PCR will produce the signal, 2^29 is a huge amplification power if you have single virus sequence after 30 cycles you will get 536,870,912 copies.

If you run RT-PCR for the virus you can determine the viral load. The earlier cycle threshold will indicate more virus copies present in the initial sample and more severe infection. For example, you have 3 patients samples and one healthy control. You got Ct values: 8, 10, 30 and 0 (no signal) for control. These results will clearly indicate that patient 1 with (ct value 8 has the most severe infection) as the sample was amplified to be detectable only after 8 cycles amplification 2^7 meaning very high initial copy number, patient 2 with ct value 15 is has a mild infection and the last patient have very few copies of the virus that was amplified only after 30 cycles 2^29. If you run PCR products on the gel you might even see these differences if they are explicit. i.e. the first patient band would be brighter. (that is the meaning of qualitative PCR). However, RT-PCR allows you to distinguish very subtle changes. I don't think it is very meaningful to use any housekeeping gene when working with the virus material so you can operate directly with the ct values. There are few techniques where you can convert ct values to the viral copy numbers, this was determined in the literature for most known viruses usually by building the linear or log curve of the known copy numbers and projecting the ct values to the curve.

I hope it helps your understanding of the PCR and RT-PCR and its use with the viruses.

real time pcr is more accurate than RT-PCR. real time pcr is quantitative and you can calculate the exact number of viruses remained after treatment.the preferred thechnique for virus identification and quantification is real time pcr

I agree to the abovementioned comments