2 replies to this topic

[medical_scientist]

Hello to anyone reading this, apologies if my questions appear to be trivial, but its better I learn now than never, (or at least get frustrated over the process of searching for info...

I am trying to design a primer for quantitative-reverse transcription PCR for the first time and am quite lost in the process of obtaining a 'cDNA', which derives my first and probably most important question. Below I will list out the questions I have to ask here. Thank you in advance for providing me any help with any of the questions.

1. What does cDNA refer to in the databases? Does it refer to (i) the sequence reflecting that of the mRNA (ii) the sequence complementary to the mRNA, which is the DNA sequence coding for the mRNA?

2. My work utilizes Drosophila as a model for neurodegenerative diseases and I have used a UAS-Gal4 system of overexpression for a certain gene. As such, in the context of PCR, I should be doing a quantitative-reverse transcription-PCR to measure for transcript levels and thereafter comparing it with the various controls such as wild-type Drosophila. Am I right about this?

3. Could a kind soul provide me a guide/process as to how he/she goes about designing a primer? (Or any books or references that can assist me in this?)

Thank you so much for any help rendered, I really appreciate it.

[Trof]

1. Can mean both, but usually it equals mRNA sequence. But for primer design you should look for reference sequence (search for your gene in Gene section in NCBI) and sequences there are called "mRNA".
Anyway unless you design reverse-transcription primers it doesn't really matter if your sequence is coding or complementary, as the primers bind on both strands.

2. Usuall control in these experiments is a "mock", transfectant with the same type of vector, but empty (no gene inserted). But maybe someone with  actual experience in these types of experiments will know better.

3. There is a thread with RT-PCR literature, good for start. You not only need primers, you need to understand the principles why you have to design primers specific, intron spanning or not, housekeeping genes primers, SYBR or Taqman with probe and so on. There is another thread about designing the primers.
A good online tool for design is primer3 or PrimerBlast, if it's not mentioned in those posts.

[medical_scientist]

Thank you so much!!!!