I am a new grad student and my project will be about miRNA. I am trying to do RT-PCR to analyze the expression of a specific miRNA in cell lines sample, but I am a little confused about how to run a good RT-PCR, and what kit I need to order to start.
As a graduate student, you will need real-world guidance from a technical expert who can give you hands-on training. Especially if you haven't done RNA work before, miRNA or otherwise, it's essential to learn RNase-free technique from someone who does this regularly with good results. Assuming you have that guidance:
1. Read up on the factors important for reliable qPCR, including http://www.ncbi.nlm.nih.gov/pubmed/19246619
2. Become familiar with different RNA isolation methods so you can determine what makes the most sense for your skills, resources, samples, and goals.
3. Since mature miRNAs are too short to amplify with traditional primers, you will need to decide whether to use a stem-loop RT primer ( http://www.ncbi.nlm.nih.gov/pubmed/16314309 ) or an adaptor ligation strategy. Be aware that there may be some biases to ligation methods, although some work quite well.
4. Realize that there's much more to learn down the road, such as choosing an appropriate normalization method.
As a graduate student, you will need real-world guidance from a technical expert who can give you hands-on training. Especially if you haven't done RNA work before, miRNA or otherwise, it's essential to learn RNase-free technique from someone who does this regularly with good results. Assuming you have that guidance:
1. Read up on the factors important for reliable qPCR, including http://www.ncbi.nlm.nih.gov/pubmed/19246619
2. Become familiar with different RNA isolation methods so you can determine what makes the most sense for your skills, resources, samples, and goals.
3. Since mature miRNAs are too short to amplify with traditional primers, you will need to decide whether to use a stem-loop RT primer ( http://www.ncbi.nlm.nih.gov/pubmed/16314309 ) or an adaptor ligation strategy. Be aware that there may be some biases to ligation methods, although some work quite well.
4. Realize that there's much more to learn down the road, such as choosing an appropriate normalization method.
- Badges
- Science topic