13 replies to this topic

[ashu2007]

Hi,

I am new in qRT-PCR. I want to do qRT PCR of two genes in normal and one stress condition. I am taking housekeeping gene GAPDH as a reference. I provided stress to bacteria while growing then isolated RNA and one is normal condition.

I have optimized conditions for RT PCR. I am able to see just one band in my RT PCR which is a desired band. I want to use comparative ct method to see the relative abundance of those genes (1& 2).

Can anyone tell me, how should I set up my experiment I mean 96 well plate?

Do I need to make any standard graph?

[pcrman]

1) you need at least triplicate wells for each sample
2) Run Gapdh and GOI for the same samples on the same plates.
3) Even though you have run regular Rt-PCR to test your primers, you still need to determine their amplification efficiency and dissociation curve before using in qPCR for your real samples.

[ashu2007]

pcrman, on 26 May 2012 - 06:04 PM, said:

1) you need at least triplicate wells for each sample
2) Run Gapdh and GOI for the same samples on the same plates.
3) Even though you have run regular Rt-PCR to test your primers, you still need to determine their amplification efficiency and dissociation curve before using in qPCR for your real samples.

Hi,

I will do that in same plate, but annealing temperature for each gene is different. Therefore I can't run them on same time.

[biznatch]

ashu2007, on 27 May 2012 - 07:41 AM, said:

I will do that in same plate, but annealing temperature for each gene is different. Therefore I can't run them on same time.

You don't have to run all your genes at the same time.  You don't even have to run them on the same day.  As long as you're consistent and proficient with your PCR set up you'll get the same results whether you run them together or seperately.  You should however keep your biologcal samples together ie. if you're comparing expression of a given gene in one normal vs one stress condition, run both that normal and stress condition for that gene in the same run.  If you're not sure just how proficient you are, then repeat some of your PCR after a few days and see if it's consistent.

Edited by biznatch, 28 May 2012 - 09:54 PM.

[ashu2007]

Hi,

Thank you for your reply. I did qRT PCR for my reference gene, but it looks weird at the starting of reaction. I have attached doc file in which I pasted my result into image form.

I am still confused about the set up of experiment. I will do them in triplicate one by one because annealing temperature is different for reference gene and two experimental gene. After that how should I proceed for comperative cT method?

Attached Files

•  New Microsoft Office Word Document.doc   237.5K   118 downloads

[biznatch]

You may be starting with too much template for such a highly expressed gene.  A dilution series (standard curve) with your Gapdh primers will tell you if/how much you need to dilute your starting DNA when measuring Gapdh.  Do you know approximately how much cDNA you're starting with?  Eg. I start with 0.5-1 ug of RNA, make 50 uL cDNA, then use 0.4 uL cDNA in each 20 uL qPCR reaction.  This works well for both high and low expressing genes.  In regards to how to set up your plates, if for example you have 3 biological replicates (6 samples altogether: 3 normal and 3 stressed), and wanted 3 technical replicates for each sample, I would set it up like this:



If you only have one biological replicate for now that's fine you can do the other ones later.  The only thing you have to repeat every time is the no template control ("blanks").

We use an Excel spreadsheet from Biorad for our ct/expression analysis, though there are probably better/easier ways to do this.  It's not the greatest (eg. your data has to be layed out perfectly before importing it) but we've been using it for years and once you get the hang of it, it's easy.  You just need to input the ct values so you can take data generated at any time from any machine.  Most qPCR machines should be able to output an excel sheet or txt file with the ct values (we use a Opticon Monitor from Biorad, also an old program).  It basically automates your calculations, including normalizing to a housekeeping gene and calculating fold change.  If you leave efficiencies at 100% you're using the ddct method, if you calculate efficiencies using a standard curve and give that data to the spreadsheet you're using the Pfaffl method (information on the two methods, at the bottom of this page).

Here's the spreadsheet:
www.bio-rad.com/LifeScience/jobs/2004/04-0684/genex.xls

and here's some instructions:
www.bio-rad.com/LifeScience/jobs/2004/04.../Manual_Rev_B.pdf

This spreadsheet shows on the left how I would organize my data before importing it into the spreadsheet from Biorad, and on the right where each value would end up once imported:
https://dl.dropbox.c...me%20setup.xlsx

Everything in the first three columns in red would be copied then imported in to the Biorad spreadsheet.  Note how each is labelled and how the coordinates are organized.

Edited by biznatch, 29 May 2012 - 10:10 AM.

[ashu2007]

biznatch, on 29 May 2012 - 09:53 AM, said:

You may be starting with too much template for such a highly expressed gene. A dilution series (standard curve) with your Gapdh primers will tell you if/how much you need to dilute your starting DNA when measuring Gapdh. Do you know approximately how much cDNA you're starting with? Eg. I start with 0.5-1 ug of RNA, make 50 uL cDNA, then use 0.4 uL cDNA in each 20 uL qPCR reaction. This works well for both high and low expressing genes. In regards to how to set up your plates, if for example you have 3 biological replicates (6 samples altogether: 3 normal and 3 stressed), and wanted 3 technical replicates for each sample, I would set it up like this: If you only have one biological replicate for now that's fine you can do the other ones later. The only thing you have to repeat every time is the no template control ("blanks"). We use an Excel spreadsheet from Biorad for our ct/expression analysis, though there are probably better/easier ways to do this. It's not the greatest (eg. your data has to be layed out perfectly before importing it) but we've been using it for years and once you get the hang of it, it's easy. You just need to input the ct values so you can take data generated at any time from any machine. Most qPCR machines should be able to output an excel sheet or txt file with the ct values (we use a Opticon Monitor from Biorad, also an old program). It basically automates your calculations, including normalizing to a housekeeping gene and calculating fold change. If you leave efficiencies at 100% you're using the ddct method, if you calculate efficiencies using a standard curve and give that data to the spreadsheet you're using the Pfaffl method (information on the two methods, at the bottom of this page). Here's the spreadsheet: www.bio-rad.com/LifeScience/jobs/2004/04-0684/genex.xls and here's some instructions: www.bio-rad.com/LifeScience/jobs/2004/04.../Manual_Rev_B.pdf This spreadsheet shows on the left how I would organize my data before importing it into the spreadsheet from Biorad, and on the right where each value would end up once imported: https://dl.dropbox.c...me%20setup.xlsx Everything in the first three columns in red would be copied then imported in to the Biorad spreadsheet. Note how each is labelled and how the coordinates are organized.

Thank you for your reply.
I am using around 1-2 ug RNA to make cDNA and I am getting 20uL of cDNA from one sample. For qRT PCR I am using 1uL of cDNA for 25uL.
My reaction mixture consist of
DD water:19uL
Taq Polymerase buffer: 2.5
dNTP: 0.5uL
F-0.5uL
R- 0.5uL
Template (cDNA): 1.0uL
Taq Poolymerase: 0.5uL
5X SYBR Greeb: 0.5uL

With the standard curve do you mean dilute those sample (cDNA) 10-1, 10-2, 10-3, 10-4?

[ashu2007]

Can it be because of I am not degrading my RNA once cDNA formed?. After making my cDNA, I am directly using that sample for qRT PCR. I am not doing RNase treatment for the cDNA formed.

[biznatch]

I think you're using too much starting material in your qPCR reaction which is why the Gapdh amplification curve looks strange.  You generally don't need to degrade the RNA after making cDNA.  I would do a 10-fold dilution series (10, 100, 1000, 10000 fold dilutions) and see what your amplification curves look like.

[ashu2007]

biznatch, on 30 May 2012 - 09:20 AM, said:

I think you're using too much starting material in your qPCR reaction which is why the Gapdh amplification curve looks strange.  You generally don't need to degrade the RNA after making cDNA.  I would do a 10-fold dilution series (10, 100, 1000, 10000 fold dilutions) and see what your amplification curves look like.

Now it is working fine, it was the problem of bubble formation during the transfer of sample from master mix to 96 well plate.
Now y question is, by using comparative cT method, I want to plot a graph for relative abundance of transcript level. How should I proceed for that?
When I was reading above article which you sent to me I understood comparative cT method, but how will I get fold for gene1 and gene2?

I am confused about this. Please help me out.

[biznatch]

The Excel spreadsheet from BioRad that I linked to will let you input ct values and will normalize to your housekeeping gene and output fold change values.

[ashu2007]

biznatch, on 04 June 2012 - 01:04 PM, said:

The Excel spreadsheet from BioRad that I linked to will let you input ct values and will normalize to your housekeeping gene and output fold change values.

Hi,

I opened that excelsheet. How will it normalize? If I am right, Is it by expressed gene- reference gene  for control and treatment?

I am unable to figure out how will I get fold change. I am really sorry, this is my first time on qRT-PCR.

[biznatch]

I think my link for the GenEx instructions didn't work.

www.bio-rad.com/LifeScience/jobs/2004/04-0684/Manual_Rev_B.pdf

These are the instructions for use the spreadsheet.

Edited by biznatch, 04 June 2012 - 01:29 PM.

[ashu2007]

biznatch, on 04 June 2012 - 01:28 PM, said:

I think my link for the GenEx instructions didn't work.

www.bio-rad.com/LifeScience/jobs/2004/04-0684/Manual_Rev_B.pdf

These are the instructions for use the spreadsheet.

Hi,

I read it, but I am unable to figure out what should I do after http://pathmicro.med...n_vs_Cycle6.htm . I got these values ddcT. Can I use those values directly for my result? If not please let me know.