Laura Leighton has explained all stages in detail, I would also suggest you to learn about validation of your method before you run any precious samples. Best wishes...
Laura Leighton has explained all stages in detail, I would also suggest you to learn about validation of your method before you run any precious samples. Best wishes...
Nanodrop (a low volume spectrophotometer) works by measuring the sample absorbance at 230nm. Absorbance at 230 is a physical property of DNA and RNA - so as you guessed, spectrophotometry can only tell you how much nucleic acid is in your sample in total, not how much of your specific RNA of interest in present.
qPCR is probably going to be the simplest and fastest way to answer your experimental question. The basic steps of qPCR are: 1) Extract RNA from tissue and quantify it 2) Convert RNA into complementary DNA (cDNA) in reverse transcription reactions 3) Dilute cDNA with water 4) Perform qPCR in technical replicates (3-4 per sample) for both your gene of interest, and a reference gene (stably expressed under your conditions) 5) Use delta-delta-cT analysis to normalise your gene of interest to the reference gene, and then express any difference between groups as fold-change.
The first thing you would need to do is identify a suitable reference gene for your species, tissue type and conditions of interest. A reference gene should be expressed at a moderate to high level, and should be stable (expression level does not change between experimental groups.) Genes encoding essential structural or metabolic proteins are often used for this purpose - eg Gapdh, Pgk1, Actb. If nobody else in your lab group does qPCR, you may have to search papers to see what reference genes are used by other groups for your species and tissue type.
You will then need to design qPCR primers for your reference gene/s, and your gene/s of interest. Many companies offer prepackaged qPCR assays for common genes and these are a great place for a beginner to start because they are already validated and you can rule out poor primer design as the cause of any problems. Before you order anything, you should read up on the difference between one-step and two-step qPCR. I recommend two-step for a beginner because there are less things that can go wrong and because you can store your cDNA to measure multiple genes from one reverse transcription. You should also be familiar with the difference between hydrolysis probe based qPCR chemistry ('Taq-Man') and SYBR green qPCR chemistry, because the reagents needed for each are not cross-compatible, and because there are some differences in troubleshooting and analysis steps. Most good molecular biology textbooks (eg, final year undergrad level) will have a good overview of qPCR, and there are lots of beginners' guides online too.
I also highly highly recommend that you get some practice tissue from the same species and ideally the same tissue type, and PRACTICE important techniques (RNA extraction, cDNA synthesis, and simple RT-PCR) several times BEFORE you do anything with samples that are precious or irreplaceable. You will absolutely need unimportant samples that you can use to check that your assays are performing as expected, so factor this into your plan from the start and you won't get caught out :)
Nanodrop (a low volume spectrophotometer) works by measuring the sample absorbance at 230nm. Absorbance at 230 is a physical property of DNA and RNA - so as you guessed, spectrophotometry can only tell you how much nucleic acid is in your sample in total, not how much of your specific RNA of interest in present.
qPCR is probably going to be the simplest and fastest way to answer your experimental question. The basic steps of qPCR are: 1) Extract RNA from tissue and quantify it 2) Convert RNA into complementary DNA (cDNA) in reverse transcription reactions 3) Dilute cDNA with water 4) Perform qPCR in technical replicates (3-4 per sample) for both your gene of interest, and a reference gene (stably expressed under your conditions) 5) Use delta-delta-cT analysis to normalise your gene of interest to the reference gene, and then express any difference between groups as fold-change.
The first thing you would need to do is identify a suitable reference gene for your species, tissue type and conditions of interest. A reference gene should be expressed at a moderate to high level, and should be stable (expression level does not change between experimental groups.) Genes encoding essential structural or metabolic proteins are often used for this purpose - eg Gapdh, Pgk1, Actb. If nobody else in your lab group does qPCR, you may have to search papers to see what reference genes are used by other groups for your species and tissue type.
You will then need to design qPCR primers for your reference gene/s, and your gene/s of interest. Many companies offer prepackaged qPCR assays for common genes and these are a great place for a beginner to start because they are already validated and you can rule out poor primer design as the cause of any problems. Before you order anything, you should read up on the difference between one-step and two-step qPCR. I recommend two-step for a beginner because there are less things that can go wrong and because you can store your cDNA to measure multiple genes from one reverse transcription. You should also be familiar with the difference between hydrolysis probe based qPCR chemistry ('Taq-Man') and SYBR green qPCR chemistry, because the reagents needed for each are not cross-compatible, and because there are some differences in troubleshooting and analysis steps. Most good molecular biology textbooks (eg, final year undergrad level) will have a good overview of qPCR, and there are lots of beginners' guides online too.
I also highly highly recommend that you get some practice tissue from the same species and ideally the same tissue type, and PRACTICE important techniques (RNA extraction, cDNA synthesis, and simple RT-PCR) several times BEFORE you do anything with samples that are precious or irreplaceable. You will absolutely need unimportant samples that you can use to check that your assays are performing as expected, so factor this into your plan from the start and you won't get caught out :)
Just isolate total RNA from the tissues and make cDNA libraries. Then design primers for your specific genes. Then you can use the prepared cDNA and Primers for a qPCR assay.
Just isolate total RNA from the tissues and make cDNA libraries. Then design primers for your specific genes. Then you can use the prepared cDNA and Primers for a qPCR assay.
Hi Omkar in addition to high quality Laura's recommandations, you should also think on arrays experiments if you need to get results on many genes. this technic will allow you to get a whole idea on genes changes in your samples with a fast and reliable way. all the best fred
Hi Omkar in addition to high quality Laura's recommandations, you should also think on arrays experiments if you need to get results on many genes. this technic will allow you to get a whole idea on genes changes in your samples with a fast and reliable way. all the best fred
Laura Leighton has explained all stages in detail, I would also suggest you to learn about validation of your method before you run any precious samples. Best wishes...
Laura Leighton has explained all stages in detail, I would also suggest you to learn about validation of your method before you run any precious samples. Best wishes...
More
VOTE
Nanodrop (a low volume spectrophotometer) works by measuring the sample absorbance at 230nm. Absorbance at 230 is a physical property of DNA and RNA - so as you guessed, spectrophotometry can only tell you how much nucleic acid is in your sample in total, not how much of your specific RNA of interest in present.
qPCR is probably going to be the simplest and fastest way to answer your experimental question. The basic steps of qPCR are:
1) Extract RNA from tissue and quantify it
2) Convert RNA into complementary DNA (cDNA) in reverse transcription reactions
3) Dilute cDNA with water
4) Perform qPCR in technical replicates (3-4 per sample) for both your gene of interest, and a reference gene (stably expressed under your conditions)
5) Use delta-delta-cT analysis to normalise your gene of interest to the reference gene, and then express any difference between groups as fold-change.
The first thing you would need to do is identify a suitable reference gene for your species, tissue type and conditions of interest. A reference gene should be expressed at a moderate to high level, and should be stable (expression level does not change between experimental groups.) Genes encoding essential structural or metabolic proteins are often used for this purpose - eg Gapdh, Pgk1, Actb. If nobody else in your lab group does qPCR, you may have to search papers to see what reference genes are used by other groups for your species and tissue type.
You will then need to design qPCR primers for your reference gene/s, and your gene/s of interest. Many companies offer prepackaged qPCR assays for common genes and these are a great place for a beginner to start because they are already validated and you can rule out poor primer design as the cause of any problems. Before you order anything, you should read up on the difference between one-step and two-step qPCR. I recommend two-step for a beginner because there are less things that can go wrong and because you can store your cDNA to measure multiple genes from one reverse transcription. You should also be familiar with the difference between hydrolysis probe based qPCR chemistry ('Taq-Man') and SYBR green qPCR chemistry, because the reagents needed for each are not cross-compatible, and because there are some differences in troubleshooting and analysis steps. Most good molecular biology textbooks (eg, final year undergrad level) will have a good overview of qPCR, and there are lots of beginners' guides online too.
I also highly highly recommend that you get some practice tissue from the same species and ideally the same tissue type, and PRACTICE important techniques (RNA extraction, cDNA synthesis, and simple RT-PCR) several times BEFORE you do anything with samples that are precious or irreplaceable. You will absolutely need unimportant samples that you can use to check that your assays are performing as expected, so factor this into your plan from the start and you won't get caught out :)
Good luck!
Nanodrop (a low volume spectrophotometer) works by measuring the sample absorbance at 230nm. Absorbance at 230 is a physical property of DNA and RNA - so as you guessed, spectrophotometry can only tell you how much nucleic acid is in your sample in total, not how much of your specific RNA of interest in present.
qPCR is probably going to be the simplest and fastest way to answer your experimental question. The basic steps of qPCR are:
1) Extract RNA from tissue and quantify it
2) Convert RNA into complementary DNA (cDNA) in reverse transcription reactions
3) Dilute cDNA with water
4) Perform qPCR in technical replicates (3-4 per sample) for both your gene of interest, and a reference gene (stably expressed under your conditions)
5) Use delta-delta-cT analysis to normalise your gene of interest to the reference gene, and then express any difference between groups as fold-change.
The first thing you would need to do is identify a suitable reference gene for your species, tissue type and conditions of interest. A reference gene should be expressed at a moderate to high level, and should be stable (expression level does not change between experimental groups.) Genes encoding essential structural or metabolic proteins are often used for this purpose - eg Gapdh, Pgk1, Actb. If nobody else in your lab group does qPCR, you may have to search papers to see what reference genes are used by other groups for your species and tissue type.
You will then need to design qPCR primers for your reference gene/s, and your gene/s of interest. Many companies offer prepackaged qPCR assays for common genes and these are a great place for a beginner to start because they are already validated and you can rule out poor primer design as the cause of any problems. Before you order anything, you should read up on the difference between one-step and two-step qPCR. I recommend two-step for a beginner because there are less things that can go wrong and because you can store your cDNA to measure multiple genes from one reverse transcription. You should also be familiar with the difference between hydrolysis probe based qPCR chemistry ('Taq-Man') and SYBR green qPCR chemistry, because the reagents needed for each are not cross-compatible, and because there are some differences in troubleshooting and analysis steps. Most good molecular biology textbooks (eg, final year undergrad level) will have a good overview of qPCR, and there are lots of beginners' guides online too.
I also highly highly recommend that you get some practice tissue from the same species and ideally the same tissue type, and PRACTICE important techniques (RNA extraction, cDNA synthesis, and simple RT-PCR) several times BEFORE you do anything with samples that are precious or irreplaceable. You will absolutely need unimportant samples that you can use to check that your assays are performing as expected, so factor this into your plan from the start and you won't get caught out :)
Good luck!
More
VOTE
Just isolate total RNA from the tissues and make cDNA libraries. Then design primers for your specific genes. Then you can use the prepared cDNA and Primers for a qPCR assay.
Just isolate total RNA from the tissues and make cDNA libraries. Then design primers for your specific genes. Then you can use the prepared cDNA and Primers for a qPCR assay.
More
VOTE
Hi Omkar
in addition to high quality Laura's recommandations, you should also think on arrays experiments if you need to get results on many genes. this technic will allow you to get a whole idea on genes changes in your samples with a fast and reliable way.
all the best
fred
Hi Omkar
in addition to high quality Laura's recommandations, you should also think on arrays experiments if you need to get results on many genes. this technic will allow you to get a whole idea on genes changes in your samples with a fast and reliable way.
all the best
fred
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