I am studying 5 microRNAs under chilling stress in tomato leaves. I am going to investigate the differential expression of these genes exposed to stress in comparison with normal conditions. I am preparing reaction solutions to analyze gene expression. In the first step, I synthesized 10 microliters of cDNA for each sample. I currently have 8 samples and I have synthesized 10 microliters of cDNA from each sample. I am currently in the process of preparing the plate. The concentration of 8 primary RNA samples is in the range of 150 to 500 ng / μl and the concentration of samples for cDNA synthesis has been reduced to 1 ng / μl. From these 8 samples, about 2 to 8 microliters (depending on the initial concentration of the samples measured by the nanodrop) were taken and added to the DNA reaction solution. The reaction mix and enzyme were added to the final solution. Due to the large number of samples, the decision has been made to reduce the volume of materials by half. As a result, the volume of final DNA created is half the amount mentioned in the kit guide (10 microliters instead of 20 microliters). We have now decided to dilute the synthesized cDNA 3 folds and use it for real-time PCR. As a result, we will have about 30 microliters of diluted cDNA. To evaluate these 8 samples, we have designed 5 microRNA-specified forward primers. The final volume of the product will be 20 microliters and the components of this solution are as follows:
One microliter of cDNA
Ten microliters of Master Mix
One microliter of the forward primer
One microliter of the universal reverse primer
Seven microliters of nuclease-free water
The standard mixes are synthesized like this:
I have added 18 microliters of nuclease-free water to 2 microliters of undiluted cDNA. Then I take 2 microliters of the diluted cDNA and added it to 18 microliters of water. I continue this serial dilution to make 1:10, 1:100, 1:1000, and 1:10000 solutions of standard mix.
The negative control and a non-template control were also used for this experiment. The following materials were used to prepare the negative control (the control without the primers) reaction:
10 microliters of Mastermix
One microliter of cDNA
9 microliters of water
The following materials were used to prepare the non-template control reaction:
10 microliters of Master Mix
One microliter Actin forward primer
One microliter Actin reverse primer
8 microliters of nuclease-free water
This test contains only one housekeeping gene which is Actin.
Do you see anything unscientific or abnormality in the process of doing this analysis?
Thank you so much for sharing your valuable experiences with me.
You should convert the same amount of RNA to cDNA from different samples. Take a negative control in which except cDNA add other components.
Before starting with your samples you should validate your assay. Determine the amplification efficiency, specificity and the dynamic range. Also confirm that bAct is a valid referece gene for your treatment. You might consider using more than one reference gene.
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