1. The reliable measure of your cDNA quality is your successful PCR. Being able to amplify fairly large fragments, providing you have no genomic DNA contamination in your sample, is a good sign of high cDNA quality. To test this you have to use housekeeping gene primers as your gene of interest expression might vary dramatically. 2. Using Real Time PCR for this reason will provide you with orders of magnitude more information. In most cases the lower Ct value the better cDNA 3. DANGER! Do not be deluded by NANODROP measurements! It measures TOTAL Nucleic acid concentration including DNA, RNA, oligos and dNTPs. So, you MUST rid off all other components before measuring cDNA. But even then, the concentration gives no information about the size of cDNA fragments which is an important measure in many cases. Size of cDNA fragments is extremely important for complete ORF amplification 4. It is VERY unlikely you can get useful information from running any gel of your cDNA prodbe. Although I have never done it, and would rather want to see these gels and/or comments on them
1. The reliable measure of your cDNA quality is your successful PCR. Being able to amplify fairly large fragments, providing you have no genomic DNA contamination in your sample, is a good sign of high cDNA quality. To test this you have to use housekeeping gene primers as your gene of interest expression might vary dramatically. 2. Using Real Time PCR for this reason will provide you with orders of magnitude more information. In most cases the lower Ct value the better cDNA 3. DANGER! Do not be deluded by NANODROP measurements! It measures TOTAL Nucleic acid concentration including DNA, RNA, oligos and dNTPs. So, you MUST rid off all other components before measuring cDNA. But even then, the concentration gives no information about the size of cDNA fragments which is an important measure in many cases. Size of cDNA fragments is extremely important for complete ORF amplification 4. It is VERY unlikely you can get useful information from running any gel of your cDNA prodbe. Although I have never done it, and would rather want to see these gels and/or comments on them
Talking about visibility of bands on gel there are two points to be taken in consideration. First, if you are running cDNA directly on gel you expect only single band. And second, any house keeping gene can be used to check the quality of cDNA, depends on primers with different product length within the same gene and then you can run the gel and look for band and copmare it with ladder on the basis of size of PCR products.
Talking about visibility of bands on gel there are two points to be taken in consideration. First, if you are running cDNA directly on gel you expect only single band. And second, any house keeping gene can be used to check the quality of cDNA, depends on primers with different product length within the same gene and then you can run the gel and look for band and copmare it with ladder on the basis of size of PCR products.
Sorry Sayan, but for the sake of good science and to avoid that others fall into this pit, I have to contradict you. UV evaluation of nucleic acids is quite useful to determine their chemical purity, but it is quite useless for quantification of specific species. Single nucleotides, oligonucleotides, as well as longer molecules all have maximal absorption around 260 nm, only the absorbance ratio A260/A280 of the single bases varies, and changes also significantly with pH between 6 and 8. Therefore, in a solution, whatever you have in - nucleotides, oligos, gDNA, cDNA, RNA - you always obtain the average ratio of the bases in the solution and you will not be able to distinguish in which proportion the different species are there. Therefore, in a cDNA reaction you will have your RNA and you will mix it with dNTPs and random hexamers and/or oligo-dT to allow the RT to produce the cDNA: the A260/A280 ratio you read at the end of the reaction you would read also at the beginning! There is no way of distinguishing if your nucleotides and hexamers have been combined into long cDNAs (which are single-stranded like the RNA) by reading UV absorbance ratio! The total absorbance might differ, due to the stacking effect, if you produce longer molecules (you might have noticed that oligonucleotides have lower specific absorbance than total RNA, which has lower specific absorbance than dsDNA...). In practice, anyway, Gennadiy is right, the easiest way to verify the quality of your cDNA in terms of integrity and full-length is to amplify a long cDNA! Best, Pietro
Sorry Sayan, but for the sake of good science and to avoid that others fall into this pit, I have to contradict you. UV evaluation of nucleic acids is quite useful to determine their chemical purity, but it is quite useless for quantification of specific species. Single nucleotides, oligonucleotides, as well as longer molecules all have maximal absorption around 260 nm, only the absorbance ratio A260/A280 of the single bases varies, and changes also significantly with pH between 6 and 8. Therefore, in a solution, whatever you have in - nucleotides, oligos, gDNA, cDNA, RNA - you always obtain the average ratio of the bases in the solution and you will not be able to distinguish in which proportion the different species are there. Therefore, in a cDNA reaction you will have your RNA and you will mix it with dNTPs and random hexamers and/or oligo-dT to allow the RT to produce the cDNA: the A260/A280 ratio you read at the end of the reaction you would read also at the beginning! There is no way of distinguishing if your nucleotides and hexamers have been combined into long cDNAs (which are single-stranded like the RNA) by reading UV absorbance ratio! The total absorbance might differ, due to the stacking effect, if you produce longer molecules (you might have noticed that oligonucleotides have lower specific absorbance than total RNA, which has lower specific absorbance than dsDNA...). In practice, anyway, Gennadiy is right, the easiest way to verify the quality of your cDNA in terms of integrity and full-length is to amplify a long cDNA! Best, Pietro
One more comment that might be at help. In order to get good quality cDNA one has to get good quality RNA first. That is where people most likely to fail. RNA is very unstable while RNA degrading enzymes are extremely stable. But there is a good news. You CAN analyze your total RNA by electrophoresis. For beginners, I would recommend to do so. Through the haze of mRNA (with no distinct bends in most cases) one will be able to see 2 distinct (although somewhat blurred ) bands representing ribosomal RNA. If you see those, you are fine to proceed to the easy part - RT reaction for your cDNA. Make sure your glassware, equipment are clean, wear gloves. Otherwise your RNA might degrade while running in gel. In case your sample is contaminated with genomic DNA, you will be able to see it on your gel. gDNA runs larger then rRNA bends, and, usually, its bend appears sharper. This is another reason to run agarose gel.
One more comment that might be at help. In order to get good quality cDNA one has to get good quality RNA first. That is where people most likely to fail. RNA is very unstable while RNA degrading enzymes are extremely stable. But there is a good news. You CAN analyze your total RNA by electrophoresis. For beginners, I would recommend to do so. Through the haze of mRNA (with no distinct bends in most cases) one will be able to see 2 distinct (although somewhat blurred ) bands representing ribosomal RNA. If you see those, you are fine to proceed to the easy part - RT reaction for your cDNA. Make sure your glassware, equipment are clean, wear gloves. Otherwise your RNA might degrade while running in gel. In case your sample is contaminated with genomic DNA, you will be able to see it on your gel. gDNA runs larger then rRNA bends, and, usually, its bend appears sharper. This is another reason to run agarose gel.
Hi, I know that this post discussed in last 3 years back. It really helps for beginner like me. Thanks for the discussion. Btw, I have one question, I think no one answered his question: how the bands will look like if we run the gel after we do regular PCR? Your answer is much appreciated. Thanks! Regards, Nisa
Hi, I know that this post discussed in last 3 years back. It really helps for beginner like me. Thanks for the discussion. Btw, I have one question, I think no one answered his question: how the bands will look like if we run the gel after we do regular PCR? Your answer is much appreciated. Thanks! Regards, Nisa
use a small aliquot of your cDNA and PCR amplify GAPDH or beta actin and if it amplifies...your cDNA is good. Be sure to add a water only neg control to be sure.
use a small aliquot of your cDNA and PCR amplify GAPDH or beta actin and if it amplifies...your cDNA is good. Be sure to add a water only neg control to be sure.
Dear Gennadiy Kovtunovych, So you are suggesting that a RT-qPCR is better than doing a conventional PCR? If you do a conventional PCR you have to run the PCR products on the gel to check for amplification of the housekeeping gene and hence the presence of cDNA in the sample but if you do a RT-qPCR reaction you don't have to run gels, you can just rely on the Ct value to confirm presence cDNA, am I right? You mentioned, "lower the Ct value, better the cDNA". How low should the Ct value be? Is there a range of Ct values that confirms good quality cDNA?
Dear Gennadiy Kovtunovych, So you are suggesting that a RT-qPCR is better than doing a conventional PCR? If you do a conventional PCR you have to run the PCR products on the gel to check for amplification of the housekeeping gene and hence the presence of cDNA in the sample but if you do a RT-qPCR reaction you don't have to run gels, you can just rely on the Ct value to confirm presence cDNA, am I right? You mentioned, "lower the Ct value, better the cDNA". How low should the Ct value be? Is there a range of Ct values that confirms good quality cDNA?
Oh, thank you very much!
Oh, thank you very much!
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VOTE
1. The reliable measure of your cDNA quality is your successful PCR. Being able to amplify fairly large fragments, providing you have no genomic DNA contamination in your sample, is a good sign of high cDNA quality. To test this you have to use housekeeping gene primers as your gene of interest expression might vary dramatically.
2. Using Real Time PCR for this reason will provide you with orders of magnitude more information. In most cases the lower Ct value the better cDNA
3. DANGER! Do not be deluded by NANODROP measurements! It measures TOTAL Nucleic acid concentration including DNA, RNA, oligos and dNTPs. So, you MUST rid off all other components before measuring cDNA. But even then, the concentration gives no information about the size of cDNA fragments which is an important measure in many cases. Size of cDNA fragments is extremely important for complete ORF amplification
4. It is VERY unlikely you can get useful information from running any gel of your cDNA prodbe. Although I have never done it, and would rather want to see these gels and/or comments on them
1. The reliable measure of your cDNA quality is your successful PCR. Being able to amplify fairly large fragments, providing you have no genomic DNA contamination in your sample, is a good sign of high cDNA quality. To test this you have to use housekeeping gene primers as your gene of interest expression might vary dramatically.
2. Using Real Time PCR for this reason will provide you with orders of magnitude more information. In most cases the lower Ct value the better cDNA
3. DANGER! Do not be deluded by NANODROP measurements! It measures TOTAL Nucleic acid concentration including DNA, RNA, oligos and dNTPs. So, you MUST rid off all other components before measuring cDNA. But even then, the concentration gives no information about the size of cDNA fragments which is an important measure in many cases. Size of cDNA fragments is extremely important for complete ORF amplification
4. It is VERY unlikely you can get useful information from running any gel of your cDNA prodbe. Although I have never done it, and would rather want to see these gels and/or comments on them
More
VOTE
Talking about visibility of bands on gel there are two points to be taken in consideration. First, if you are running cDNA directly on gel you expect only single band. And second, any house keeping gene can be used to check the quality of cDNA, depends on primers with different product length within the same gene and then you can run the gel and look for band and copmare it with ladder on the basis of size of PCR products.
Talking about visibility of bands on gel there are two points to be taken in consideration. First, if you are running cDNA directly on gel you expect only single band. And second, any house keeping gene can be used to check the quality of cDNA, depends on primers with different product length within the same gene and then you can run the gel and look for band and copmare it with ladder on the basis of size of PCR products.
More
VOTE
Hi Nisa,
First of all he did not ask how PCR "fragments" look on a gel.
In most applications one suppose to see one DNA band of a predicted size
Hi Nisa,
First of all he did not ask how PCR "fragments" look on a gel.
In most applications one suppose to see one DNA band of a predicted size
More
VOTE
Sorry Sayan, but for the sake of good science and to avoid that others fall into this pit, I have to contradict you.
UV evaluation of nucleic acids is quite useful to determine their chemical purity, but it is quite useless for quantification of specific species. Single nucleotides, oligonucleotides, as well as longer molecules all have maximal absorption around 260 nm, only the absorbance ratio A260/A280 of the single bases varies, and changes also significantly with pH between 6 and 8. Therefore, in a solution, whatever you have in - nucleotides, oligos, gDNA, cDNA, RNA - you always obtain the average ratio of the bases in the solution and you will not be able to distinguish in which proportion the different species are there.
Therefore, in a cDNA reaction you will have your RNA and you will mix it with dNTPs and random hexamers and/or oligo-dT to allow the RT to produce the cDNA: the A260/A280 ratio you read at the end of the reaction you would read also at the beginning! There is no way of distinguishing if your nucleotides and hexamers have been combined into long cDNAs (which are single-stranded like the RNA) by reading UV absorbance ratio! The total absorbance might differ, due to the stacking effect, if you produce longer molecules (you might have noticed that oligonucleotides have lower specific absorbance than total RNA, which has lower specific absorbance than dsDNA...).
In practice, anyway, Gennadiy is right, the easiest way to verify the quality of your cDNA in terms of integrity and full-length is to amplify a long cDNA!
Best, Pietro
Sorry Sayan, but for the sake of good science and to avoid that others fall into this pit, I have to contradict you.
UV evaluation of nucleic acids is quite useful to determine their chemical purity, but it is quite useless for quantification of specific species. Single nucleotides, oligonucleotides, as well as longer molecules all have maximal absorption around 260 nm, only the absorbance ratio A260/A280 of the single bases varies, and changes also significantly with pH between 6 and 8. Therefore, in a solution, whatever you have in - nucleotides, oligos, gDNA, cDNA, RNA - you always obtain the average ratio of the bases in the solution and you will not be able to distinguish in which proportion the different species are there.
Therefore, in a cDNA reaction you will have your RNA and you will mix it with dNTPs and random hexamers and/or oligo-dT to allow the RT to produce the cDNA: the A260/A280 ratio you read at the end of the reaction you would read also at the beginning! There is no way of distinguishing if your nucleotides and hexamers have been combined into long cDNAs (which are single-stranded like the RNA) by reading UV absorbance ratio! The total absorbance might differ, due to the stacking effect, if you produce longer molecules (you might have noticed that oligonucleotides have lower specific absorbance than total RNA, which has lower specific absorbance than dsDNA...).
In practice, anyway, Gennadiy is right, the easiest way to verify the quality of your cDNA in terms of integrity and full-length is to amplify a long cDNA!
Best, Pietro
More
VOTE
One more comment that might be at help.
In order to get good quality cDNA one has to get good quality RNA first. That is where people most likely to fail. RNA is very unstable while RNA degrading enzymes are extremely stable. But there is a good news. You CAN analyze your total RNA by electrophoresis. For beginners, I would recommend to do so.
Through the haze of mRNA (with no distinct bends in most cases) one will be able to see 2 distinct (although somewhat blurred ) bands representing ribosomal RNA. If you see those, you are fine to proceed to the easy part - RT reaction for your cDNA. Make sure your glassware, equipment are clean, wear gloves. Otherwise your RNA might degrade while running in gel.
In case your sample is contaminated with genomic DNA, you will be able to see it on your gel. gDNA runs larger then rRNA bends, and, usually, its bend appears sharper. This is another reason to run agarose gel.
One more comment that might be at help.
In order to get good quality cDNA one has to get good quality RNA first. That is where people most likely to fail. RNA is very unstable while RNA degrading enzymes are extremely stable. But there is a good news. You CAN analyze your total RNA by electrophoresis. For beginners, I would recommend to do so.
Through the haze of mRNA (with no distinct bends in most cases) one will be able to see 2 distinct (although somewhat blurred ) bands representing ribosomal RNA. If you see those, you are fine to proceed to the easy part - RT reaction for your cDNA. Make sure your glassware, equipment are clean, wear gloves. Otherwise your RNA might degrade while running in gel.
In case your sample is contaminated with genomic DNA, you will be able to see it on your gel. gDNA runs larger then rRNA bends, and, usually, its bend appears sharper. This is another reason to run agarose gel.
More
VOTE
Hi, I know that this post discussed in last 3 years back. It really helps for beginner like me. Thanks for the discussion. Btw, I have one question, I think no one answered his question: how the bands will look like if we run the gel after we do regular PCR? Your answer is much appreciated. Thanks!
Regards,
Nisa
Hi, I know that this post discussed in last 3 years back. It really helps for beginner like me. Thanks for the discussion. Btw, I have one question, I think no one answered his question: how the bands will look like if we run the gel after we do regular PCR? Your answer is much appreciated. Thanks!
Regards,
Nisa
More
VOTE
You can check by nano, its easy and best method. Usually 1.7 to 1.8 of 260/280 value for cDNA is considered as pure and can be used for PCR or qPCR.
You can check by nano, its easy and best method. Usually 1.7 to 1.8 of 260/280 value for cDNA is considered as pure and can be used for PCR or qPCR.
More
VOTE
The best way to check your cDNA quality is to do PCR using refrence gene and the primer of your target gene. Once you get results then your cDNA is ok
The best way to check your cDNA quality is to do PCR using refrence gene and the primer of your target gene. Once you get results then your cDNA is ok
More
VOTE
use a small aliquot of your cDNA and PCR amplify GAPDH or beta actin and if it amplifies...your cDNA is good. Be sure to add a water only neg control to be sure.
use a small aliquot of your cDNA and PCR amplify GAPDH or beta actin and if it amplifies...your cDNA is good. Be sure to add a water only neg control to be sure.
More
VOTE
PCR with house-keeping genes followed by running the PCR products on agarose gel.
PCR with house-keeping genes followed by running the PCR products on agarose gel.
More
VOTE
Dear Gennadiy Kovtunovych,
So you are suggesting that a RT-qPCR is better than doing a conventional PCR?
If you do a conventional PCR you have to run the PCR products on the gel to check for amplification of the housekeeping gene and hence the presence of cDNA in the sample but if you do a RT-qPCR reaction you don't have to run gels, you can just rely on the Ct value to confirm presence cDNA, am I right?
You mentioned, "lower the Ct value, better the cDNA". How low should the Ct value be? Is there a range of Ct values that confirms good quality cDNA?
Thank you very much.
Dear Gennadiy Kovtunovych,
So you are suggesting that a RT-qPCR is better than doing a conventional PCR?
If you do a conventional PCR you have to run the PCR products on the gel to check for amplification of the housekeeping gene and hence the presence of cDNA in the sample but if you do a RT-qPCR reaction you don't have to run gels, you can just rely on the Ct value to confirm presence cDNA, am I right?
You mentioned, "lower the Ct value, better the cDNA". How low should the Ct value be? Is there a range of Ct values that confirms good quality cDNA?
Thank you very much.
More
VOTE