Hi, thank you, Martina Pigoni, for your reply. Yup, i am interested to study the target gene expression. I am like in the maze that the variation in amount of template for qPCR will give different Ct value, that is why i am thinking to "standardize" the amount of template loaded into qPCR, from either amount of RNA or amount of cDNA.

Thanks.

spec and normalise mRNA before RT reaction, then co amplify a housekeeping gene that is represented by the same copy number og that of your gene of interest within the gemome use the house keeping gene as a normalisation factor

Hi,

According to my experience, efficiency of qRT PCR depends on many factors like primer quality, RNA quality, Sybr green reagent or Reverse transcriptase quality. If any one of the factors are problematic, successful qRT PCR is not possible. However, considering all the factors are in good quality, I can suggest from my personal experience that prepare cDNA using 1-1.5 micrograms of total RNA in 20 micro-liter of reaction volume. From this reaction mixture, use 2 micro-liter as template and standerise your primer in different temperature and different MgCL2 concentration. I think, in this way, your problem might be solved.

Hi Martin Devonshire, can i know how should i "normalize" mRNA? The RNA extracted is total RNA through Phenol-Chloroform method.

Thanks to Dr. Martina Pigoni, i think i get what you mean by using a housekeeping gene as comparison and normalize factor for the target gene expression. The outcome we obtained from qPCR is just the relative ratio, right? So, even we got the variation in cDNA concentration, it will also give variation in both target gene and housekeeping expression, but in the linear relationship, right? Yet, we can still obtain the Cq value of target gene by normalize it with housekeeping gene, right? Please correct me if i am wrong.

Thanks to Guru Prasad Maiti for your input.

Apologies, the "m" was a typo on my part.. had been typing mRNA a lot that day.. quantify and normalize the extracted RNA

Hi Tzu shan Ng.

Looking you gel I see the following:

lane a, c d, you have something in the load region that can be a big DNA, ou protein with DNA, In lane B you did not see this huge band . If you treat RNA sample with DNAse and loaded in a , b, c , d lanes, problably in lane B your DNAse worked because you not see any DNA, that you clearly see in others . now if you compare lanbe b and E you can se a very similar profile, that can make you think. does de DNAse worked?

so you need to improve your extration, because some inhibitor are presente in your samples, as someone comment can be phenol, chroloform? so you can try to use a fresh reagentes, ou a kits (QIGen work fine) .

My concern is this RNA can be used to RT PCR quantification? or just to ampyfy a specific cDNA? you must check DNAse, and your extraction reagents and also run the gel before and after DNAse treatment......

I think if you will make real time pcr for quantification your results will be not confidente......to interpret your experimental idea.......

Must use 2100 system from Agilent or neww system called tape station.....best regards.....