As show in attachment, same like this how can I find it?

Thanks. I have few questions

1. What is purpose of this method?

2. we can use RNA directly instead of cdna?

3. If expression of RNA or cdna not good or very faint so can we use it?

4. Which kit you prefer to use and how many cycles?

Hi Bushra,

1. Tubulin is a house-keeping gene. Researchers use it as a reference gene or an "internal control" for quantitative RT-PCR (qRT-PCR). It is  necessary for researchers to normalize the expression levels of target genes (i.e. genes-of-interest) using suitable reference genes to ensure reproducible and accurate quantitative expression measures.

2. Except Tubulin, there are other house-keeping genes, which researchers can use as reference genes. I attach 2 papers for you about different reference genes used in Plants study and Animal study. In the papers, it describes what will be a good reference gene, and which reference genes are more reliable than other reference genes in using for a specific study (yellow highlights).

3. I am trying to explain the concept of using 'internal control' to you with a more relaxed way, by using your attached picture as an example and dialogues between two people. See below:

*Professor: Your gel looks good (see attachment). Gene expression for AeSPL6 in your gel seems to decrease steadily, while gene AeSPL9 seems to increase steadily from day 0 to day 24.

*Bushra: Thanks.

*Professor: But, how do you know this is real? Can it be possible that the change was due to human errors? Such as, less amount of total RNA you used towards day 24, leading it to the 'decreased' RT-PCR bands on gel?

*Bushra: Oh, no. The amount of total RNA for each sample in each lane used for qRT-PCR was added equally.

*Professor: How do you know that you added equally? Do you have an evidence/proof for that?

*Bushra: Yes, look at the last entry (at the bottom of the picture), I used tubulin (a house-keeping gene) as an internal control for each sample. And all the band intensities were equally presented from qRT-PCR results. If due to man-made errors, the band intensity would not have been the same.

*Professor:  ...........

For your question #2: Can we use RNA directly, instead of cDNA?

1. People either use total RNA or mRNAs (most likely, yours is total RNA) to make cDNA, and use cDNA for PCR, in order to complete the whole RT-PCR procedure.

2. Without RNA, you cannot produce cDNA, and without cDNA, you cannot have RT-PCR result. So, both total RNA and cDNA are important ingredients and needed in completing RT-PCR procedure.

For your question #3: If expression of RNA or cDNA not good or very faint so can we use it?

You have to remember: Unlike house-keeping genes, some genes only produce mRNA at certain tissues and certain stages of plant development. So, for these genes, if you want to isolate RNA from them, you have to isolate it at a particular tissue and stage (age of the plants) to ensure that the mRNA of your gene-of-interest (GOI) is there. If the total RNA you isolate did not contain the specific mRNA of your GOI, you will not have a product (band) from RT-PCR. This is one of the reasons that you can have a RT-PCR result without a band.

thank you soo much Yuan-Yeu Yau for very clear and detail answer 

By the way, if you want to use Tubulin as your internal control (reference gene), you will need the primers to amplify Tubulin of the plants you are working.

You might be able to use the primers listed in the paper I attached earlier. I will attach again. Since DNA sequences of some house-keeping genes are very conserved, so the same primers used for one plant species can be used for the other species. So, get the Tubulin primers listed in the paper and use them to amplify the gDNA or cDNA of your plants to see whether they work (page 3-4, 'TUA' = Tubulin gene).

@Yuan-Yeu Yau here in protocol they mention add 10 ul mercaptoethanol per 1 ml buffer. Buffer is around 18 ml, so it means we add 180 ul mercaptoethanol to 18 ml buffer?

1. Correct, 180 ul, Bushra. Per = 'for each'.

2. By the way, when you add 2-mercaptoethanol, add it in a venting hood, not on the working bench. It is not good for your own health and other people's health in the lab.

I attach its material safety data sheet (MSDS) for you (see the yellow highlights).

"The substance is toxic to the nervous system, mucous membranes. The substance may be toxic to upper respiratory tract, eyes, central nervous system (CNS). Repeated or prolonged exposure to the substance can produce target organs damage. Repeated exposure to a highly toxic material may produce general deterioration of health by an accumulation in one or many human organs."

3. Let me know if you have more questions.