Oligonucleotide Probes - Science topic

It is bit required to know about exactly what you want to see and experimental part? May be the labeling is not perfect or some quenching is going on or experimental error!!! This is preliminary thought, but need to know what exactly you want and what is the output?

Also check https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/apurinic-site

Hello, see the info attached may be useful for you.

Hi Parama Gandi

I'm sure you'll have enough in this paper coming from a specialized workshop:

stay safe

fred

okay, thank you once again!

The design of primers verse probes can both be down fairly easily with the use of the companies design tools (as long as you know the regions you want to target) and most companies will provide you with assistance.

Usually most people prefer probe based methods over primer methods since they work better of degraded samples (i.e. you need enough intact copies of your regions to get amplification). If you have good high molecular weight DNA and little to no degradation both primer and probe methods should be find. If you have degraded samples I would suggest using probe methods. I tend to just go with probe methods so that I can use them on both degraded sample (i.e. environmental DNA) and intact samples (i.e. fresh tissue extracts).

Hope this help and good luck!

Hello , see info attached may be useful for you. Good luck!

thanks a lot

I suggest ligation of a 3' end neutral synthetic fragment such as one having the C3 spacer at the 3' end before the main ligation, so you will have a 3' blocked fragment.  

@Björn Abendroth thank you for answer!

1. I use oligonucleotide probe, so i ordered cy3 labeled oligo. So I don't label for my oligo.

2. It was solved when I use wet kimtech(wet by DW), and do not stacks.. during hybridization, water can enter between slide and coverslip?

3. I spot oligos by hand, and use arrayit superamine2. spotting is okay except after baking, it remains white donught..

Hello,

What is the commercial source for these oligo probes and did they work. 

Thanks 

Hi Matthias,

I was looking for yeast specific FISH probes. I came across this information which I think may be suitable for you.

Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures. Kempf VA, Trebesius K, Autenrieth IB. Journal of clinical microbiology. 2000.

Probes Sequence

5'- CTC TGG CTT CAC CCT ATT C -3'

Please go through the publication and find out if it is helpful.

Regards

Riyaz

unspecific binding, steric trapping, or naturally the hybridication = so that is, of course, one thing you might need to consider if you did not see it...

but if you want to see how oligos distribute as oligos, so not for FISH, then you should do this in vivo - send me an email, and let's get in contact, but you might consider the following papers for that:

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging. Weidemann T, Wachsmuth M, Knoch TA, Müller G, Waldeck W, Langowski J. J Mol Biol. 2003 Nov 21;334(2):229-40.

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Wachsmuth M, Weidemann T, Müller G, Hoffmann-Rohrer UW, Knoch TA, Waldeck W, Langowski J. Biophys J. 2003 May;84(5):3353-63.

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin. Tóth KF, Knoch TA, Wachsmuth M, Frank-Stöhr M, Stöhr M, Bacher CP, Müller G, Rippe K. J Cell Sci. 2004 Aug 15;117(Pt 18):4277-87. Epub 2004 Aug 3.

Detection of NGF-receptors TrkA and p75NTR in human tumor cell lines and effect of NGF on the growth characteristic of the UT-7/EPO cell line. Westphal G, van den Berg-Stein S, Braun K, Knoch TA, Dümmerling M, Langowski J, Debus J, Friedrich E. J Exp Clin Cancer Res. 2002 Jun;21(2):255-67.

so and for high resolution stuff and quantification:

Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei. Rauch J, Knoch TA, Solovei I, Teller K, Stein S, Buiting K, Horsthemke B, Langowski J, Cremer T, Hausmann M, Cremer C. Differentiation. 2008 Jan;76(1):66-82. Epub 2007 Nov 26.

The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions. Jhunjhunwala S, van Zelm MC, Peak MM, Cutchin S, Riblet R, van Dongen JJ, Grosveld FG, Knoch TA, Murre C. Cell. 2008 Apr 18;133(2):265-79. doi: 10.1016/j.cell.2008.03.024.

Super-resolution imaging reveals three-dimensional folding dynamics of the β-globin locus upon gene activation. van de Corput MP, de Boer E, Knoch TA, van Cappellen WA, Quintanilla A, Ferrand L, Grosveld FG. J Cell Sci. 2012 Oct 1;125(Pt 19):4630-9. doi: 10.1242/jcs.108522. Epub 2012 Jul 5.

@Ambadas B Rode, Unfortunately the paper is behind a pay wall and even with my university credentials, I can't access it for free.  I noticed that you are the first author.  Would you be willing to give me a copy of the paper?

The classic example of NWC base pairing is Hoogsteen pairing (https://en.wikipedia.org/wiki/Hoogsteen_base_pair). In essence, ration around the C1-N bond between the deoxyribose ring and the base can permit base pairing using alternative hydrogen bonding patterns. Mismatches occur when one base pairs with what is generally considered to not be its cognate base, for example a G-G base pair. Both NWC base pairing and mismatches are generally energetically unfavorable compared to WC pairing or pairing with a proper cognate base. However, this penalty can be small enough that mismatches can be tolerated in DNA (the bonding energy of neighboring bases permits base pairing around the mismatch), or the formation of NWC base pairs in unusual circumstances (e.g. repetitive tracts of G-rich sequence, the enzymatic sites of certain bypass polmerases, etc.). Additionally, certain modifications to base pairs can alter their base pairing activities (e.g. guanine oxidation, https://en.wikipedia.org/wiki/8-Oxoguanine), which can end up producing mismatches following DNA replication.

Hi Hans,

Thanks for the reply. I had already used the Stellaris FISH probe from biosearch and it was very good. We manage to identify one of the isoform mRNA. We tried with the other isoform but no luck. When both mRNA was evaluated with multiple alignment method, they has 60% difference of sequence. However, we did not obtain any reading from the second isoform. either the cells did not have the second isoform mRNA or our flow cytometer unable to detect the signals. We do test both FITC and CalFluor for signal and no problem. Inthe end,we confirmed one of the isoform and manage to proceed other experiment with the results. Attach is one of the paper we publish and hope you can provide some insight on the results.

Supershift is sometimes tricky - antibody can affect probe-protein formation. Did you try various competitors to track the exact site of binding? I am using set of unlabeled competitors - each with one or two mismatches (when compared to the probe). 

Alternatively, think about another factor - eg Egr1 often competes with Sp1.

thank you very much. I have noticed also that the elution of the streptavidin changes if I use or not Biotin or a biotynilated molecule. so is it possible that a biotynilated molecule/biotyn stabilizes the streptavidin from the leakeage?

I hope this paper can help you

W. Haiss et al., Determination of Size and Concentration of Gold Nanoparticles from UV-VIS Spectra,

Anal. Chem. 2007, 79, 4215-4221

Dear Anukana,

One reason could be that your oligos might undergo dimerization upon long storage. so, be confirmed that whether your oligos are single stranded. Second point is that inclusion of Mg++ ions, if it is insufficient, it could lead to failure in binding.

All the best wishes,

Regards

Rajkumar

We have done FISH in Tetrahymena thermophila using telomeric oligos. See Loidl and Scherthan, 2004, attached. Let me know if you need a more detailed protocol.

I guess that would be all right. If Isolated RNA means that total RNA from cell or tissue, You will be able to observe that downregulation of target RNA level by qRT-PCR. If you want to observe specific mRNA(Bcl-2, GAPDH, ACTB......) in vitro, you should use T7 RNA polymerase.

Hello Michael,

I have been doing RNA FISH successfully for several years.  I have not worked with neuronal cells but have worked with ESCs.  First of all, try using COT-1 DNA in your hybridization buffer to quench hybridization of repetitive sequences.  (Is it possible that your scramble probe has a repetitive sequence?). Secondly, I wash my slides 1X 50% Formamide @ ~40° C. In preparing my slides, I pretreat with an extraction step of  ice cold CSK buffer/0.5% TX-100/RVC for 10 minutes.  I then fix my slides with 4% paraformaldehyde.  I store them @ -20° C in 70% ethanol until needed.  I have found that my slides work have less background if I let them age for 1-2days prior to hybridization. I have attached a complete protocol for your reference.   

There is a picture of Xist RNA FISH on my personal page showing a typical signal.  

It is not clear from your question whether you need RNA oligonucleotides (so the oligo has an RNA backbone) or DNA oligonucleotides that are antisense to an RNA sequence!

Adding a non-template Poly(A) tail to RNA can be achieved by Poly(A) polymerase and ATP in the presence of buffer and Mn. I buy mine as a Poly(A) Tailing Kit from Ambion/Life Technologies AM1350.

Thank you very much for the replies. It helped me a lot.

Although the star strand of the mature miRNA sequence is now known not to be discarded (probably playing roles), the guide strand (here the 5p-strand) is the major miRNA species and plays more important functions in the cells. I recommend you to order a probe that detects the guide strand of let-7a (here: let-7a-5p).

Hi Alexandre,

During my days in academia, I synthesized oligos using both Expedite 8909 and the ABI 394. We used basic spectrophotometric readings to determine the concentration of our oligos and this seemed to work fine. With this in mind, I would favor measurements using the Nanodrop. The Nanodrop can provide a scan of the quality of the oligo which should be a Gaussian curve with a peak at 260 nm. However, there are two issues to consider when measuring absorbance with the Nanodrop.

First, what media will the oligos be measured in, water or buffer? If they are in water then the Nanodrop can also be blanked with water. But if they are in buffer, you may need to likewise blank the Nanodrop with the same buffer for more accurate results.

The second issue is the concentration of the oligos. Spectrometric readings whether using the nanodrop or any other spectrometric instrument has a limited range. You may need to dilute the oligos, then make an additional dilution in order to get a more accurate reading. You could make the final dilution in water and avoid the need to blank with buffer. I would recommend that the absorbance values be limited to 0.2 to 0.8 for the best accuracy.

There are two strategy for DNA probe immobilization on iron ferum oxide nanoparticles. First step is SAM interaction where u can treated your nanoparticles with APTMS or APTES through silanization process to introduce Amines Group. Then, this amines modified electrode is modify with glutaradehyde solution. With activation of EDC/NHC, u can easily immobilize your probe DNA. Second step, is electrostatic interaction. Same procedure above when your nanoparticle treated with aptms, aptes. Since Amine group is positive charge and DNA has negative charge phosphate bond, it can bind each other. I hope I can answer your question.

Because you know, there are 3 bonds between G and C

when there are only two bond between A and T so if the ratio was much it will affect the process of ligation and melting of the primer and will need high temperature may adversely affect the process of DNA replication.

Normally people use ready to use chemistry from various supplier, but from your "Q" I felt as if you are mixing all component separately and doing QPCR with taqman probe. As far the Taq polymerase concern you need normal Taq polymerase to do Q-PCR. You cann't use the Taq polymerase which has proof reading activitiy because such taq even degrade your taqman probe before even PCR start. Normal Taq only have 5'-3' polymerase which you actually required for Taqman probe activity while proof reading polymerase has both 5'-3' and 3'-5' exonuclease activity.

One of the way I used for having TAR RNA that is a hairpin was to warm it at 90 °C during 2 min in HEPES buffer or else and put it directly in ice for 15 min. This protocol promote the formation of the monomolecular complex.

Yes, concentration can matter, but it doesn't always.

So, when you have two things binding to each other, the association (binding event) is bimolecular and depends on the concentrations of the two components. The amount of binding is determined by the component in excess (this is sometimes counterintuitive - the maximum physical number of bound molecules is of course determined by the molecule in trace, but the percent bound is determined by the component in excess). If one or both of the components is saturating (say 5-10 times greater than the Kd), then you should be all set and happy.

For dissociation, the reaction is unimoleclar (one thing falling apart), so it does not depend on concentration at all.

So, if your primer/target interaction is extremely stable, the concentrations of target and primer can affect how much of your target is bound, but whatever is bound should essentially stay bound during the time of your experiment.

If your primer/target interaction is very weak and dissociates quickly, you can favor binding by having a high concentration of primer to drive rebinding. This means at equilibrium, you would have more bound.

You can modify the stability of your interaction by changing salt concentrations too. Many Tm calculators will ask you the concentration of your primer and the salt concentration to give you a better estimate of the Tm. Try oligoanalyzer from IDT for example. Make sure you specify whether you are using DNA or RNA as DNA/DNA < DNA/RNA < RNA/RNA in terms of stability.

Does that make sense?

Taq amn probes can be ordered with something other that FAM, like TET- 521. 536 (excitaion-emisson), JOE-520. 548 or VIC. ~555(emission). What machine are you using, atleast 3 filters should do your work. Generally ROX or TAMRA are used for internal control so your probe-primer should have a different label that these two.

Dr. Ulrike, even if GFP is tagged and its a fusion construct, then GFP along with the miRNA will be transcribed and should be a single fusion miRNA, why should it be in the protein? If its not an IRES GFP where GFP will be expressed separate as opposed to a fusion construct, is there a problem of getting a fusion RNA?

So any miRNA which is made will have a GFP tag along with the sequence of the gene/miRNA of interest.

Please correct me if I am missing something.

http://www.ncbi.nlm.nih.gov/pubmed/20554852 I am not sure if the link is very helpful.

Besides LNAs carboxyfluorescein labeled MO oligos were also recently used to detect miRNA, so that could be another way. (http://bio.biologists.org/content/early/2012/04/11/bio.2012810.full) But miRNAs can be pretty abundant, so I do think the same limitations as I described before would apply.