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Individuals who carry the wildtype genotype for SNPA also carry the wildtype genotype for SNPB.
Individuals who carry the heterozygous genotype for SNPA also carry the heterozygous genotype for SNPB.
Individuals who carry the mutant genotype for SNPA also carry the mutant genotype for SNPB.
It is pretty obvious that the two polymorphisms are in complete linkage disequilibrium, right?
So the D' should equal to 1.
But, how do I calculate that?
I found a web-based software which seems to be easy to use, but I don't know what number should I key in onto each cell --> http://www.oege.org/software/cubex/
Now supposed I have 1000 wildtype, 800 heterozygous and 600 mutants for both polymorphisms, what number shall I put in the cells?
I tried the following (based on the frequencies of my genotypes):
But I got a D' value of -0.011 instead of 1.
Can anyone help?
You may recommend other softwares or simple methods for calculating the D' and r2.
Thanks in advance.
Due to shortage of time (I need to do some trial tests today), I proceeded with DNA and RNA isolation with the reagents added with denatured ethanol.
The spectrophotometric readings were good - the yield and purity were comparable to DNA/RNA isolated with the reagents with non-denatured ethanol.
Let's hope any impurities that may be present would not interfere with downstream applications.
I used RNeasy.
What method did you use for the extraction? An abnormally high A260 reading could be an indication of Phenol contamination, giving you the unusually high A260/A280 ratio.
I wonder how that particular sample become contaminated with phenol when the other 15 were fine.
And I was told that RNeasy spin columns could effectively remove phenol.
15 of the samples had A260/280 ratio between 1.9 and 3.5. I think the range was still acceptable although 3.5 is actually quite high.
But...the remaining one sample had an A260/280 ratio of about 30!
I checked with 2 spectrophotometers and both gave me the same reading.
What does this absurdly high A260/280 ratio mean?
I added denatured ethanol to all the necessary buffers, then only I came to realize that we should use non-denatured ethanol for molecular biology applications because denatured ethanol contains impurities.
What should I do now?
Can I proceed with DNA and RNA isolation using the reagents?
If not, my supervisor is going to kill me OMG!!!
Wow, thanks for the reply after so long.
Cells from cancer tend to get into blood stream so you can get the specific genes that are normally expressed only in one type of tissue and overexpressed in cancerous tissue from blood. But you need normally expressed gene as well for reference.
Then how about RNA from saliva?
But I have read somewhere that if we isolate RNA with Trizol, the RNA integrity tends to be lower than isolating with RNeasy kit.
In our lab, we have observed that with kits that use spin columns is normal to loose the smaller RNAs (they are not retained in the column), while those are still there with Trizol.
Back to your picture, to me it looks like you do have some degradation, also may have some DNA contamination (top of the gel, still in the well). It also looks like the gel is maybe overloaded and what I think is degradation (smeary bit between distinct bands) might just be due to too much RNA.
So if I need both small RNA and high integrity RNA for my work, based on your experience, would you suggest me to use Trizol or RNeasy?
I loaded 4ul RNA into the gel, and I only came to know later that for RNA, 2ul is sufficient, so most probably the smear is due to overloading of RNA.
But I'll run gel electrophoresis again after this.
DNA contamination is not unexpected because I did not use DNase this time (because this is only a trial experiment of mine).
I have failed several times in the past for my RNA isolation, and this is the first time I succeeded.
It turned out that my previous failed attempts was due to insufficient grinding of the tissues.
Thanks for the reply.
Degradation presents as a smear. Bands are other rRNA bands
But I have seen other people's RNA gel image, and most of them got something like this (only 2 clear bands without those smaller bands):
So why are those other rRNA bands present only in my samples?
Does the presence of those other rRNA bands indicate something good or something bad?
I isolated RNA from human tissue samples using RNeasy.
I electrophoresed the RNA obtained and this is what I got:
P.S. 100bp DNA ladder was used instead of RNA ladder because we don't have RNA ladder in our lab.
From the gel image, two clear bands can be seen (supposed to be 18S and 28S rRNA).
But there are also some smaller bands down there.
Does this indicate RNA degradation or something else?
P.S. The tissue samples were preserved in RNAlater for 1 month before RNA isolation.
P.S. We don't have Bioanalyzer in the lab, so I cannot analyse it in the instrument.
P.S. I did not check the purity and concentration of the RNA because the only spectrophotometer in our lab has been send for repair.
Oh..so if the protocol recommends us to resuspend the cell pellet into 200ul of buffer, and I have an initial cell suspension volume of 200ul, does it mean that I don't have to perform the centrifugation and resuspension?
It would be so that you can concentrate your cells into a much smaller volume to facilitate the DNA extraction.
May I know what is the purpose of obtaining this cell pellet?
Can we just vortex the suspension and use it directly without pelleting the cells?
Well, they are easiest to collect...
Also just found this via google http://iranpath.org/...71209000055.pdf , a review where they also discuss, starting from p.7, the similarities between breast tissue and the salivary glands and the respective fluids, although it seems to focus on proteins rather than RNA...
Oh, thanks for the reply.
Anyone else could tell me why RNA from blood/saliva is used instead of RNA from tissue?
Way too expensive...
any of the PLOS group? Don't know about publishing costs though.
- PLOS Biology US$2900
- PLOS Medicine US$2900
- PLOS Computational Biology
- PLOS Genetics US$2250
- PLOS Pathogens US$2250
- PLOS Neglected Tropical Diseases
- PLOS ONE US$1350
Anyone has other recommendation?
So in many studies, researchers analyze the RNA expressions of certain genes in affected tissues and compare them to matched normal tissues.
For example, some breast cancer researchers would investigate the expression of BRCA1 in cancerous and noncancerous breast tissue, so that they can know whether deregulated BRCA1 expression occurs in breast cancer.
These are what I understand from my undergraduate course.
But, recently I came across quite a lot of studies, which use blood or salivary RNA to study gene expression of tissue-specific diseases such as breast cancer.
I fail to figure out the rationale of using blood or salivary RNA instead of RNA from the tissue to study gene expression.
Could someone please enlighten me on this?
I am looking for a journal
- with an ISI impact factor of around 2.0 - 2.5
- with low or no publication fee (my advisor is not willing to sponsor any amount greater than USD 200)
- in the area of medical science/cancer genetics/cancer molecular biology/colorectal cancer or other related areas
Did you run a No template control along with this PCR? I am just guessing if these smears are directly from your template DNA. What is the source of DNA?
To check the primers you can reduce the primer concentration too e.g. down to 0.1 uM (you have now a concentration of 0.25 uM if you have 20 ul total volume per tube). If the smear becomes less it's perhaps a primer-dimer problem (they can also be amplified, if they overlap in a way that the polymerase can attach; this can happen with badly designed primers).
And try out AmeyaP's idea of course.
Thanks for the suggestions. Will try them and let u know the result.
The band of interest was there, but the primer dimers are soooooo much more pronounced than the band of interest.
I have also tried (1) touchdown pcr, (2) increasing the annealing temp, (3) adjusting (increasing/decreasing) the duration of denaturation, annealing, extension, but all gave me similar results.
Can anyone advise?
The band size of interest is 200bp and I got a lot of smear (should i call it smear because it has specific discrete pattern).
Here the details of the PCR:
2X PCR master mix ---> 10 ul
Forward primer (10 pmol) ---> 0.5 ul
Reverse primer (10 pmol) ---> 0.5 ul
Template DNA ---> 0.5 ul
I have tried diluting the template DNA, but I still get all those smears (i.e. when I diluted the template, both the band of interest and the smear become faint)
I don't think the problem lies on the primers, because
(1) there has been many previous publications utilizing this primer pair
(2) i thought probably my primers degraded or something like that, so I tried to order new sets of primer (same sequence, but newly synthesized one) from different companies
Initial denaturation (95°C, 2 minutes)
Denaturation (94°C, 30 seconds)
Primer annealing (60°C for 45 seconds)
Elongation (72°C, 1 minute)
Final extension (72°C, 5 minutes)
Can anyone help? Thanks in advance~
I loaded 3ul.
How much of your PCR volume did you load on the gel? It looks very overloaded to me. The smearing below your product could be primer dimer.
Just out of interest, can I ask what the rationale for using those cycle conditions is? It seems odd to me to have two annealing temperatures that are SO close.
60'C is the highest achievable temp. I mean, after 60'C, i can no longer get any pcr product. so i chose to use tat one as the starting temperature.
and i chose 59'C because from my optimization experiments, 59'C can result in multiple bands, so i would not go below that temp.
there should be no problem with primer design. i have BLASTed and have checked with a few softwares. Moreover this is the primer pair obtained from literature. A lot of people have used this primer pair.
Given the 98 denature, I assume you are using Phusion. Phusion master mixes are usually 2x master mixes. You should double check to make sure you are not missing the added water.
Yes, it's Phusion. It was a typographical error. What I used was 10ul master mix plus 8.5ul water.
98'C 30 sec initial denaturation
98'C 5 sec denaturation
60'C 5 sec annealing
72'C 5 sec extension
98'C 5 sec denaturation
59'C 5 sec annealing
72'C 5 sec extension
72'C 2 min final extension
my band of interest is 200bp
i got the band
but there are smearing below the band:
untitled.bmp 468.18KB 1387 downloads
what has gone wrong and how should i rectify the problem?
FYI, my pcr master mix contain 18.5ul master mix, 0.5 ul forward primer, 0.5 ul reverse primer, 0.5 ul DNA.