Nephrite

Hello.
I make some efforts to describe change in expression of few genes in a time-course study. My experiment comprises one control and few experimental groups.
So far I`ve normalized the expression of each target gene for each single sample vs the ref. gene in all groups of the study. I used the dCt method and I collected groups of dCt data.
Then I determined the ratio of expression in the control group and in the test groups.
What I am supposed to do now?
Would it be enough if I plot the means of dCt of each group (control and test groups) with CI, SD or SEM bars and compute the P values between the groups, i.e. one-way ANOVA comparing more than two means with the appropriate multiple comparisons test?
I am reading a paper now: Statistical analysis of real-time PCR data. Yuan JS et al., BMS Bioinformatics 2006, 7:85.
I thing they recommend some SAS program and they discuss the use of the Wilcoxon test, which is for two groups of data.
I have some experience with the GraphPad Prism.
Isn`t it good enough for this job?

Hello.

I wonder if anybody can help me with an advise because I really need it.....

My case - I finished MS in Molecular biology, I lost 3-4 years in my home Institution (by this time the local politic was still very supressive toward the young generations), then I used the first opportunity to leave my country, I established a colaboration with one professor abroad, after some time I started PhD and 4 years later I defended.

In between, my professor offered me 1 year postdoc in his University, which I accepted and I worked in several different topics.

Now I just returned to my country.

To my big unfortune all these years I was completely alone professionally - I was learning alone, I was building research hypotheses alone, I was working alone, I was interpreting results alone, I was writing papers alone, I was fighting with oponents alone, etc.
I mean, it happened that I didn`t work in a group, so all ideas of my professor were completely new for both of us and I was the only one to develop them.

In result, I feel extremly tired of research.
Exhausted.
Empty.
So many nights, so many years thinking only if the hypothesis is correct, how to prove it, how to build the philosophy of the work, how to perform the experiments, how to explain the results, how to motivate the work - I can`t anymore......
In addition, life in other country was sad, I was the only one foreigner in the Institution.
In addition, I feel a bit useless for the society because of the abstract nature of my work.

So, two weeks ago, before I left my professor announced me as one of the very best pupils he has ever had - this should mean that I can provide something useful.
I can deal relatively well with few techniques (immunohistochemistry, WB, PCR and related) and I have some competences.
We (the professor and I) managed to make some papers and if we are lucky we will publish three more works.
But that`s all.
I feel like an educated and qualified but useless member of the society.

Can anybody give me some clues what else I can work? How to be more useful for the society, for myself and for my future family? Is there anything routine and practical that people like me can work? For sure I don`t want to continue with research (to be honest, if this is the only one option in front of me, then I prefer to clean houses) but probably there should be something else that I can do?

Any advise would be highly appreciated.

Hello.

The task is to follow expression of four genes (incl. the housekeeping) in six experimental groups (three individuals each).

This makes 76 single ractions (all samples (3) from all groups (6) with all primers (4) + 4NTC).

I was told that two real time plates can not be compared but I wonder how the statistical evaluation would be done?

Which variant would be better:

1. One plate with 76 single reactions and some.....calbration between them (I heard some trick with the treshold line) - three-four times;

OR

2. One plate with 1 individual from each group with all primers, in triplicate - three times, for all individuals;

OR

3. One plate with all samples fom all groups (18) and only one primer, in triplicate - four times for the four primers?

Thank you

I feel ashamed of this idiotic post but nobody has ever teached me this and I need to elucidate it once forever:

1. Molar concentration reffers to g/L (i.e.one FW (g/mol) in 1 liter), and hence milimolar is mg/mL, micromolar in ug/uL, etc. - Yes or No?

2. When we measure concentration and the mashine gives a dimension ug/mL but our stock solution is in microliters, then we convert the result in ng/uL - Yes or No? In the same logic, if recommended concentration of something is 50 ug/mL but the volume of reaction is in uL we apply 50 ng/uL - Yes or No?

3. The capital ''M'' in the dimension uM is reffered to Liter and means ug/L - Yes or No? So, if recommended concentration of something is 1 uM (1 ug/L) but volume of reaction is in microliters then we apply 1 picogram/uL of this reagent - Yes or No?

I would highly appreaciate if somebody dedicate some amount of time and control this fo me.

Thank you.

Hello.

My RNAs are extracted from 10 paraffin embeded tissue sections/sample. I used the Qiagen kit with columns. The amount of RNA in all samples is app. 40-50 ng/ul in 20 ul volume.

A reviewer recommended two different approaches to check if there is contaminating gDNA. One of the approaches is to treat my RNA samples with RNase and then to measure for gDNA.

I intend to measure it before and afer treatment, but I don`t know how to deactivate the enzyme. In the instruction manual (Fermentas) they say that heating of samples will not deactivate the RNase and they suggest chloroform/phenol extraction.
However, my sample is RNA predominantly, in little amount and small volume.

Do I really have to perform this extraction step (which I don`t know how in this small volume)?

Can I try to denature the enzyme with some low concentration of......SDS or something? Will it interfere with the NK?

Do I really should deactivate this enzyme just to perfom 10 min measurement? I will not use these samples after that.

Thank you.