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J092's Content

There have been 9 items by J092 (Search limited from 27-January 19)


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#177001 How to make the most of masters year?

Posted by J092 on 24 October 2015 - 07:19 AM in Journal Discussion - How to Get Papers Published

Hi, I am applying to a masters and beginning at the start of 2016. I have written and designed my project(in addition to keep adding details and creating action plans). I really do wish to get my masters paper published, are there any tips on how to make the most of masters year the best it can be?

I am not progressing to phd immediately as I simply cannot afford to stay there right now so only focusing on masters project (its masters in research degree). Are there any tips to get my content posted in a high science journal? (I have looked extensively but also very helpful to get input of those who have achieved what I wish to)

Any help is appreciated.
Thank you



#176547 E-Cadherin Expression in OAW-42 Cells

Posted by J092 on 27 August 2015 - 04:01 AM in Cell Biology

I have not been able to confirm this but this is something I will be doing in my own project in due time.

 

I was wondering how E-cadherin can still be expressed in this cell line? (known from research), in my previous project I did do, I found the knockdown of CLDN3 to downregulate Twist and found there was an association and expected E-cadherin to be decreased. However, in another paper SNAIL & SLUG are not expressed in OAW-42 cells and therefore is predicted to maintain E-cadherin's expression, I'm really confused on how this can be, if anyone can kindly suggest any literature to dig into or give a helpful response, will really appreciate it.

 

Thank you




#173483 Difference between Ct Expression and Up/DownRegulation?

Posted by J092 on 21 February 2015 - 03:36 PM in PCR, RT-PCR and Real-Time PCR

I've done my analysis on my Ct values. Firstly when I checked if there was downregulation or upregulation (I wanted downregulation as I was performing gene silencing) I used the Livak method, no problems. So for one experiment I got 0.166 so downregulation as I wanted, that's fine.

 

However, to be able to do statistical analysis and compare my results, I calculated my ratios (2^Ct values) for each sample separatley normalised to the controls of the target & reference gene (means) and got a value of 0.1828.

 

What I don't understand is why are these two values different? Am I not looking for the same thing which is the rate at which downregulation occured? And if so why is it two different values?

 

What I do follow is that when you're trying to find whether it's been silenced or not, you look at the overall values together and that confirms whether it has or not...my main question is what is the value in which it has been deregulated as I've one from Livak method using the means of Ct values and then another method which provides average ratios




#173269 Protein knockdown method?

Posted by J092 on 07 February 2015 - 04:05 PM in siRNA, microRNA and RNAi

I performed siRNA transfection on cells monitoring mRNA expression, how would I relate this to determining a change in protein expression?

Is it only through Western Blotting, there's no way I can tell if mRNA levels affected protein expression?



#173229 t-test & qRT-PCR

Posted by J092 on 05 February 2015 - 08:30 AM in PCR, RT-PCR and Real-Time PCR

Double-post by accident, apologies.

 

 

merged them

hobglobin

Attached Thumbnails

  • table2.png



#173228 t-test & qRT-PCR

Posted by J092 on 05 February 2015 - 08:29 AM in PCR, RT-PCR and Real-Time PCR

I've been reading a lot of how to analyse qRT-PCR results. So far I have calculated:

-Delta Delta Ct - therefore know whether my target gene is up/down-regulated

 

I was looking at other documents online which split the target and controls up by repeats in a table and calculate everything separately (compared to the method of finding the mean of the Ct values and working through it). I wish to perform the t-test on my data but am not sure how to do so as some sources say that you:

 

1) Write your experiments are separate values

2) Subsequently calculate means

3) Average delta delta Ct values & perform t-test on those

 

An example of my trouble in reference to the attached image is:

1) When I calculated the Delta Delta Ct value for the whole experiment, I got the same answer as the Target treatment which is 0.046, but no idea how to get 1.002 and even their ΔΔCq
Expression for Non-Targeting Controls do not match: (2^-0.1 is different - 0.960 & 0.945??)

 

My main aim is from my data to be able to:

1) Calculate delta delta Ct value (Done)

2) Standard Deviation - can do if I do everything separately

3) t-test

 

If anyone has any help for steps two and three - would appreciate it. thanks.




#173024 Sirna transfection DNA contamination

Posted by J092 on 28 January 2015 - 01:48 PM in siRNA, microRNA and RNAi

I've done sirna transfection on my cells but when run the products on an electrophoresis gel it was shown to be highly contaminated with DNA, they were treated with DNase I, I then did cDNA and checked if silencing worked using qRT-PCR, it did not. However I had kept the conditions in which the first time it had worked with other samples. Is the DNA contamination a possible reason for it not working even if treated with DNase I?



#172873 Claudin 3 Annealing Temperature

Posted by J092 on 24 January 2015 - 01:25 AM in General Lab Techniques

I'm really sorry if this is wrong place but can't find information anywhere!

 

Primer sequence

FP: GTCCGTCCGTCCGTCCG

RP: GCCCAGCACGGCCAGC

 

I did bench top PCR at 54 degrees, not so good then reduced to 52 degrees for  qRT-PCR and have to adjust again to get it correct and go even lower. I've been looking at papers and one website suggested 60 which seems way too high...




#172872 Can you use same samples for real time pcr 2nd time?

Posted by J092 on 24 January 2015 - 12:04 AM in PCR, RT-PCR and Real-Time PCR

Hi guys, I made samples yesterday and ran through the real time pcr machine, I've got my original samples as in my CDMA, primers etc. I can make it all from scratch. However I'm a bit confused the real time pcr tubes I put into the machine - am I able to use those again to repeat my real time for second time? I would think not as sample has been degraded, annealed etc. so would I have to do everything from scratch? I would like to confirm so I can plan my day, thank you.




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