Experimental set up for qRT PCR - (May/26/2012 )

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?

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.

pcrman on Sun May 27 02:04:44 2012 said:

ashu2007 on Sun May 27 15:41:16 2012 said:

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?

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.

biznatch on Tue May 29 17:53:51 2012 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.

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.

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.

biznatch on Wed May 30 17:20:47 2012 said: