3 replies to this topic

[ColoSarah]

This is my first go at real time RT-PCR, so I have tons of questions. I designed primers for my gene of interest. I have run my primers with a One-step SYBR green kit and done a melt curve to look for any non-specific products. I have also done a temperature gradient with the SYBR green kit for the annealing temperature to see where I get the lowest Ct value, highest fluorescence and correct PCR product melting temp. I just ordered my probe and these are where my questions come in...

I'm doing a relative gene expression assay, so I need to find "reference" genes (3 or more), design primers and determine if they are expressed relatively the same over my experimental conditions and various tissues? Can anyone give me any advice as to how to go about finding good reference genes and testing them? I know there are the commonly used ones including, GAPDH, beta actin, etc. but I've done a bunch of reading and they aren't necessarily as great as people have always thought. I suppose I could start with those...

One-step vs. Two-step PCR? I have decided to go with the two-step, but I don't know if that is necessarily the best choice or if it even matters. I figured since I'm doing a relative gene expression assay I want to use cDNA from the same pool for my GOI and reference. They can be run in separate wells, yes? I think multiplexing might make my head explode.  

I also need to run a standard curve to determine the efficiency of my PCR reaction. Can I just dilute my cDNA 2 or 10 fold, run the PCR and generate the curve? Should this be done after my primers and probes have been optimized together or with SYBR green?

I really just need a start to finish protocol on how I should go about designing my whole thing. It's tough when this is all new to you and no one in your lab is a molecular biologist.

Any input would be so greatly appreciated! Thanks!

[chrisbelle]

ColoSarah, on Apr 17 2010, 03:18 AM, said:

1. This is my first go at real time RT-PCR, so I have tons of questions. I designed primers for my gene of interest. I have run my primers with a One-step SYBR green kit and done a melt curve to look for any non-specific products. I have also done a temperature gradient with the SYBR green kit for the annealing temperature to see where I get the lowest Ct value, highest fluorescence and correct PCR product melting temp. I just ordered my probe and these are where my questions come in...

2. I'm doing a relative gene expression assay, so I need to find "reference" genes (3 or more), design primers and determine if they are expressed relatively the same over my experimental conditions and various tissues? Can anyone give me any advice as to how to go about finding good reference genes and testing them? I know there are the commonly used ones including, GAPDH, beta actin, etc. but I've done a bunch of reading and they aren't necessarily as great as people have always thought. I suppose I could start with those...

3. One-step vs. Two-step PCR? I have decided to go with the two-step, but I don't know if that is necessarily the best choice or if it even matters. I figured since I'm doing a relative gene expression assay I want to use cDNA from the same pool for my GOI and reference. They can be run in separate wells, yes? I think multiplexing might make my head explode.  

4. I also need to run a standard curve to determine the efficiency of my PCR reaction. Can I just dilute my cDNA 2 or 10 fold, run the PCR and generate the curve? Should this be done after my primers and probes have been optimized together or with SYBR green?

5. I really just need a start to finish protocol on how I should go about designing my whole thing. It's tough when this is all new to you and no one in your lab is a molecular biologist.

Any input would be so greatly appreciated! Thanks!

Dear Sarah,

Refer numbering as above:
1.Probe??? are you using SYBR Green or probe? what kind of probe?
2. refer to journals or previous studies on the recommended reference genes for your organism, tissue/cell or treatment type.
3. Yes 2-step is fine. and you can run separate genes in separate wells.
4. You can dilute your cDNA 2,5,10 or whatever fold you wish. You can also use any other cDNA as long as its from the same organism/tissue/cell. use your standard curve period to do optimizations. if you are using probe, why the SYBR Green???
5. You should read up before starting anything, not after you have decided the chemistries, method, etc... Obviously since you have ordered the probe/primers/etc you already 'designed' your experiment. I learned real-time from scratch by reading manuals, journals and the internet. It's not that big deal.

Chris

[ColoSarah]

chrisbelle, on Apr 22 2010, 03:00 AM, said:

ColoSarah, on Apr 17 2010, 03:18 AM, said:

1. This is my first go at real time RT-PCR, so I have tons of questions. I designed primers for my gene of interest. I have run my primers with a One-step SYBR green kit and done a melt curve to look for any non-specific products. I have also done a temperature gradient with the SYBR green kit for the annealing temperature to see where I get the lowest Ct value, highest fluorescence and correct PCR product melting temp. I just ordered my probe and these are where my questions come in...

2. I'm doing a relative gene expression assay, so I need to find "reference" genes (3 or more), design primers and determine if they are expressed relatively the same over my experimental conditions and various tissues? Can anyone give me any advice as to how to go about finding good reference genes and testing them? I know there are the commonly used ones including, GAPDH, beta actin, etc. but I've done a bunch of reading and they aren't necessarily as great as people have always thought. I suppose I could start with those...

3. One-step vs. Two-step PCR? I have decided to go with the two-step, but I don't know if that is necessarily the best choice or if it even matters. I figured since I'm doing a relative gene expression assay I want to use cDNA from the same pool for my GOI and reference. They can be run in separate wells, yes? I think multiplexing might make my head explode.  

4. I also need to run a standard curve to determine the efficiency of my PCR reaction. Can I just dilute my cDNA 2 or 10 fold, run the PCR and generate the curve? Should this be done after my primers and probes have been optimized together or with SYBR green?

5. I really just need a start to finish protocol on how I should go about designing my whole thing. It's tough when this is all new to you and no one in your lab is a molecular biologist.

Any input would be so greatly appreciated! Thanks!

Dear Sarah,

Refer numbering as above:
1.Probe??? are you using SYBR Green or probe? what kind of probe?
2. refer to journals or previous studies on the recommended reference genes for your organism, tissue/cell or treatment type.
3. Yes 2-step is fine. and you can run separate genes in separate wells.
4. You can dilute your cDNA 2,5,10 or whatever fold you wish. You can also use any other cDNA as long as its from the same organism/tissue/cell. use your standard curve period to do optimizations. if you are using probe, why the SYBR Green???
5. You should read up before starting anything, not after you have decided the chemistries, method, etc... Obviously since you have ordered the probe/primers/etc you already 'designed' your experiment. I learned real-time from scratch by reading manuals, journals and the internet. It's not that big deal.

Chris

I used a one-step SYBR green kit to optimize my primers. I did a melt curve at the end of my PCR to look for non-specific products and to make sure my melting temperatures matched up with that of my amplicon.

When I run my standard curve with my diluted cDNA I will want to run it with my probe, yes? I will have to optimize my primer and probe concentration I imagine.

[Trof]

You have to run your standard curve with the same system you will use for your samples. If you'll analyse the samples with a probe assay you run the standard curve also with a probe. You do this after you have optimised the assay and use the same reaction conditions as with your samples.