How to design Real time PCR primers?

How to probe deeply into plant stress resistance? Does plant adversity research eventually come down to the molecular level? Have many confusions about this topic, and dont know what can i focus on if i want to further investigate plant stress resistance? My master research is about physiology, transcriptome and metabolome changes of plant under stress. I am interested in this topic, but i feel like i can't catch a point. Because there are many related areas in this topic.

I want to prepare the three levels of PEG-6000, which are 5% (mild drought), 10% (moderate drought), and 20% (severe drought). Many people said that to make 5% of PEG-6000, we need to take 5g of PEG-6000, dissolve it in water, and make a volume of 100 ml. In the same way, 10g for 10% PEG and 20g for 20% PEG. I want to confirm if this calculation is correct. The second question is how to apply this solution in potted plants grown in vermiculite culture? Is there any specific dose (5ml or 10ml per pot)? Most of the papers do not mention the dose per plant. your answers to my question will be highly appreciated. Thanks

I am doing differential gene expression analysis using transcriptome data. Many papers report the PCR amplification of certain genes after gene expression analysis. My question is, how important is PCR amplification after gene expression analysis? Can I publish my work without this step? Any specific gene is not my target, I want to see what gene expression changes. Any suggestions will be appreciated.

Thanks

Mousumi Mary

Hi there. I have some data from RNA-seq experiments. In PCA, we observe that the dominating patterns in our data are explained by other variables, rather than by those we used to classify our samples. Hence, our predefined groups are not visible in the PCA representation (they are mixed up). However, in the cluster heatmap, samples are clustered according to our variable, and is clear that the expression vectors (the columns of the heatmap) for samples within the same cluster are much more similar than expression vectors for samples from different clusters.

Given that in ALL the heatmaps cluster samples correctly according to our variable, is it possible that PCA is filtering out information that is meaningful to our samples?

How should I interpret these findings?

Thank you

I used OligoPrimer, PrimerBlast and PrimerQuest. Shouldn't the results be the same?

Recently I did RNA seq on mycobacterium. I got the TPM data from one of our colleagues in the Bioinformatics department because they helped us to analyze the raw data.

Then, I'm interested in making a volcano plot from the data. Do you think it's possible to get a p-value from TPM data?

I have been asked to submit a paper on a special issue of Genes- MDPI. The impact factor of the journal is showing to be 3.4 but I saw conflicting articles about the MDPI journals being a predatory one. Though the editor of the issue is a reputed person in the field, I am bit confused about the journal in general. What are your thoughts?

I was preparing a phosphate buffer and adjusting its pH to 7.2 from 9.0 by adding monobasic phosphate. In the beginning, it started to decrease fast but then, it went slowly (it was about pH=7.6). Even though I added too much monobasic solution, its pH was not increased easily. Then, it went to pH=7.3 easily but it stopped at pH=7.3 again. I wonder why it happened. I know it is related to its titration and the titration graph is fine to understand but I need a comprehensive explanation for it.

Iam currently working on my paper regarding the genetic diversity. I want to visualize my distance matrix data on a heat map, and I think it will be good as it is new for me. Is there anyone here can suggest me a software or online tools that I can use to generate a heat map to visualize distance matrix?

Thank you