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- Oleg V Moskvin added an answer in RNAseq:How to perform RNAseq statistics?
RNAseq delivers gene expression data from experiments with several biological replicates for two or more conditions. Once you have the processed gene expression data comparisons between conditions can be performed and the genes selected should be weighted for the probability that they appear differentially expressed by chance. Which approaches to do that are there? Is Significance Analysis of Microarray (SAM) the right choice?
Please keep in mind that with RNA-Seq, statistical test for differential expression should be performed at count level (NOT using measures that are derived after normalization by gene length such as FPKM/RPKM/TPM). Counts, of course, need to be normalized between libraries, and to our experience, simple median normalization works the best (we did a systematic study involving many factors -http://biorxiv.org/content/early/2014/10/17/010488 ). In the end of the day, you will be interested in a biological story, i.e. functional patterns of cellular response. Counterintuitive result of our above-mentioned study (that involved assessment of 6,912 combinations of technical and biological factors) was very low (close to zero, indeed) impact of low-level processing stage and lower than expected impact of the choice of DE testing method on the derived "functional signature of response". At the same time, choice of geneses enrichment method was critical.Following
- Hypertetraploid cell line, is possible to do gene editing?
Hi to all! I'm entering the field and so I have a lot of question..
The first one is the model: We are currently working with a cell line that is Hypertetraployd , and I'm wondering if it could be possible to do HDR on this cell lines. As I understand, the system does not have an allelic preference. So, in your opinion could be possible to use this system or I have to switch to another model? Another conceptual question: How the Cas9 recognize preferentially a ssOD instead of the other allele? or it is not? so the frequency of mutation would be quite low?
many thanks for any input....
they did it in wheat see doi:10.1038/nbt.2969 nature biotechnology
simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew
Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes1, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator–like effector nuclease (TALEN)2,3 and clustered, regularly interspaced,
short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins6. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations7. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks
caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.Following
- Could somebody recommend a good working selective growing medium for Glomus intraradices and Trichoderma harzianum?
Could somebody recommend a good working selective growing medium for Glomus intraradices and Trichoderma harzianum?
Glomus intraradices..now Rhizophagus.. is a symbiont microrganism..so, I guess, the only good working media could be a root of a plant from a sterile colture.Following
- What is the minimum size of tensile test specimen according to ASTM? I want to fabricate one dye for powder compaction and samples for tensile test. What is the minimum size of tensile test specimen so that a smaller dye can be made according to specimen?
Gage length=25 mm, Width=6 mm, Radius of fillet=6 mm, Overall length=100 mm.
Some time fixture can also be used to hold the specimenFollowing
- How can you design a system that can detect and amplify frequencies in the 8Hz - 12Hz range ?
I'm specifically trying to measure alpha brainwave. Is there any article, paper, publications available on this subject that could be recommended? Thanks!
Hi Emmanuel! As underlined by Alexander, the most apt way to do that depends on your purpose.
Are you interested in online detection or offline measurement? Depending on that, you might be needing to balance computation accuracy and time efficiency.
Below you find the reference to a solid general review on alpha activity, I hope it might help youFollowing
- Is it possible to decompose ammonia gas by UV Radiation?
Ammonia gas has Lambda Max between 212-215 nm. Is it possible to decompose Ammonia using Ultraviolet radiation? If it is possible, how much (power) watt will be required to decompose one liter of ammonia in one minute? How we can increase the rate of decomposition of ammonia?Following
- What is the incubation time required to use insulin as an inducer to study its signalling pathway in skeletal muscle (C2C12 and HSMM)?
Please can anyone suggest the incubation time required for Insulin for studying the downstream phosphorylation of its substrate proteins of mouse and human skeletal muscle (C2C12 and HSMM)?
it depends on what is the result or function you focused on.Following
- How can some bacteria affect fish health?
The following colonies of bacteria were found in our mesocosms :
Does anyone know if these bacteria can affect fish health. I have found literature on Lysinibacillus sphaericus but not the other microorganisms.
May be you can ask to Stéphane Betoulle, Reims University - France ?
- Before adding the restriction sites, my primers have a 9°C Tm difference but after adding them, the Tm values narrowed. How does this affect my PCR?
The primers that I designed initially had Tm values of 54.26°C (Fwd primer) and 45.53°C (Rev primer) before I added the restriction sites. After I did that, however, I managed to get the Tm adjusted to 68.59°C and 69.54°C for both the forward and reverse primers respectively. This was done using the Primer-Blast tool. I do realize that the difference in the Tm value is quite large, but I'm not sure whether this will severely affect the PCR.
Would I have to set a lower annealing temperature for a few cycles first before increasing it to the Ta for my complete primers (including the restriction sites)?
Do the restriction sites you added actually bind to template? If not you have to omitt the sequence of the RE site from the Tm calculation, so your original temperatures are the correct ones. 45 degrees is on the low side though...Following
- Is anyone familiar with the Hazard Ratio model?
Hi. When odds ratio from logistic regression have large confidence interval for data from cross-sectional study, it is suggested to use Cox Proportion Hazard Regression Models with constant time at risk and report hazard ratios as hazard ratios have a small confidence limit. Is it good to use the hazard regression model ? Thanks
You can solve your problem using log-binomial regression because it can obtain RR in cross-sectional study and has a small standard error. Of course, you also use Cox hazard ratio model to obtain RR of a factor when time is fixed in 'one'.Following
- What is the reason of high K2O and low total Fe2O3?
We analyzed 8 samples of alterated jurassic basalts. Analysis results of major oxides are attached to this question as a word document.
What is the reason of high K2O and low total Fe2O3?
The question is not clear. In your data the Fe2O3 range from 6.05 to 14.41 wt%, which is not realy low and the K2O range from 0.17 to 6.39 wt, which display for some samples a very low K2O contents. Are the basalt samples from the same location?
Generally the high K2O content and low Fe2O3 content in the basic rocks is a result of fractionation processes of magma and degree of partial melting from the mantle.Following
- Why the Western blot for SCN1a (+200 kD) is not working anymore with 5M Urea buffer brain proteins extract?
I am running western blot to detect transmembrane protein of >200 kDa (SCN1a). I extracted my Proteins with 5M Urea Buffer, and I keep them at 4*C. I run Western using Tris-Acetate gradient gels 3-8 % with Tris acetate running buffer (Invitrogen). I prepare my samples in Laemli buffer (BioRad) or Sample buffer (from Invitrogen) with BetaMercapto or LDS as reducing agents. I do not boil the samples. I use wet transfer for 2h at 30V.
The westerns worked very well, and suddenly from one day to another it stooped. First progressively my high MW band that before were very strong after 5 min (in the cassette) started to fade, then my Low MW, and by the end using the same samples I was unable to detect any band after few hours!
Please give me any possibilities what the problem is. I tried to resolve the technical problem by changing the lot of gels, running buffers, Laemli, secondary and primary antibodies, electrophoresis and transfer systems...
And the last test my friend performed for me using hers and mine samples, and all samples looked ok on PonceauS but the blot worked only on her samples - meaning there is something wrong with mine!
I need to add that these samples are VERY important and impossible to reproduce (as it is a human material).
I will be very thankful for any advices!
Have you seen that your sample turn precipitated? Some proteins precipitate after frozen. If yes, it could affect to your protein concentration, so to your loading amount. I would suggest that you can run SDS PAGE again and load double than before. When you detect signal on blot, first try the same substrate that you used before; if you don't see signal, wash the blot several time and try the more sensitive detection kit (Femto kit from Pierce or other brand).Following
- How does the reference electrode effect the corrosion rate measurement?
Some people use, in three cell electrode, electrodes made from the same material? For example carbon steel is used as working, counter and reference electrodes rather than using carbon steel as a working electrode and using Ag/AgCl as counter and reference electrodes. How does this effect the corrosion measurement? Will the corrosion rate be the same in both cases?
You should work with a Hydrogen reference electrode. That reference does not influence the electrolyte. All other reference will poison the electrokyte with Potasstium, Chloride, Silver or Mercury ions (of course in small quantities, but detectable)Following
- How are competencies of Mayors in other parts of the world regulated towards public police?
Public police is in Belgium the first and most important provider of surveillance and control (and registration) of social disorder in the public domain. I am searching for sholars publishing on the same topic in other countries, EU and not EU to join my network. Is surveillance and control in the public domain on petty crime and social disorder pluralised or not? Are tendencies towards plural policing wide spread in Europe?
- If I want to make an emulsion, can I mix two surfactants with a similar HLB (i.e. HLB~4 with HLB~1.8)? Will this result in any complications?
Note that these HLB values show greater affinity towards an oil phase relative to an aqueous phase
Agreed witha all above, there should be no problem, but, as pointed out in the previous reply, it is always best to check! Very likely the surfactant mixture may give you an improvement in performance, or possibly you can work with less surfactant loading.Following
- Why does high carbon steel behave in a brittle manner?
It may have a very high yield stress, but fracture occurs at an elongation of few percent.
It depends also on the heat treatment. If high carbon steel is annealed, then it can become less brittle than if quenched. But, in the case it is annealed, the higher % of cementite will lead to less ductility than in low carbon steel. If high carbon steel is quenched, then martensite and other metastable structures may be formed. These structures are very hard, thus rendering material brittle.Following
- Is Optical Density value at midlog of bacterial growth curve the same as with the addition of an antibiotic?
Should the highest level of RNA expression should be on log phase?
To compare the difference of RNA expression, can I use OD value of a cell at the midlog of bacterial growth curve to extract RNA from bacteria with and without an antibiotic?
Is the Optical Density value at midlog of bacterial growth curve the same as the one plus an antibiotic.
Another question is, I can see the log phase of bacterial growth curve (semilog) but for bacteria with antibiotic, the line is not linear (semilog) like log phase, can I use the same method to determine OD at center of the non-linear.
If this is incorrect could someone share research paper, protocol or free software for growth curve (equation of log phase, OD at log phase). I will appreciate your help
Thank you in advance
(sorry, I'm not good in English)
If the antibiotic you added are effectively inhibiting the growth of the corresponding microorganisms at the time point you measure the OD, I guess you would see differences between the OD value of with and without antibiotic. Furthermore, I doubt if the overall level of RNA expression is highest during log phase. It depends on which genes you are going to measure it transcription level. For example, stress-response related genes might not be expressed very high during log phase, but at the onset of stationary phase and/or when abrupt environmental changes are sensed. Good luck for your research!Following
- How can I calculate ppm of analyte gas based on flow rate ratio?
Is it possible for me to calculate the ppm of the targeted gas based on its flow rate ratio with the carrier gas. For example, if I flow ethylene gas at 1 sl/m mix with Nitrogen flow at 50 sl/m into the test chamber of 1 liter cubic capacity, what will the ethylene ppm inside the chamber? Will flow duration effecting the ppm level inside the chamber?
PPM is another measure of concentration. Gases are quite simple to calculate concentrations with, as you can assume, for the most part, that molar concentrations are the same as volumetric concentrations. From the example you've provided, the concentration of ethylene gas is equal to the flowrate of ethylene over the total flowrate (one divided by fifty one). To turn this into PPM, simply multiply this ratio by one million, but i'd suggest PPM is not the best way to report this concentration.Following
- Who creates value in the fashion luxury sector?
Customers find value in rational, emotional or symbolic aspects of luxury items, but when asked very often refer to superior quality, outstanding materials and craftsmanship (which is a rational type of value). Do luxury fashion brands still reflect these values or are they just efficiently communicated?
Who, in a luxury fashion value chain is responsible for value adding? Where does it lie? In production or in a communication sphere? Is it designed maybe for effective distribution?
thanks a lot, that's another VERY HOT topic worth exploration... new distribution channels that add value to the items...but at the same time they can dissapoint the more old - fashioned customers...Following
- How to preserve the anaerobic bacteria?
The preservation of the anaerobic bacteria in skimmed milk broth.
using 15% glycerol in suitable medium is best way to preserve for longer periods.Following
- Is there any research about the lifetime of a company? The survival rate for a company after 1, 2, 3, ... years.
No Brasil o SEBRAE acompanha taxa de Mortalidade de Empresas. O Último relatório pode ser acessado no site:
- How to enhance climate change resilience and sustainability in urban areas?
I agree with Professor Rajiv Pandey. Life style adjustment, coupled with infrastructural support, is certainly an option to enhance climate change resilience and sustainability in urban areas. To stimulate life style adjustment, it is important making people aware. Customized research is desirable in this regard.Following
- What are the best practical methods for measuring water flow in agricultural field?
However, there are known methods, but farmers are not understood. We need a simple method that farmers are able to practice it.
Dear Ali Reza,
depends on the amount of water / discharge:
Installing a weir would mean that the measurement for the farmers would be a water-level measurement, e.g., using a yard-stick. It would, however, also require determination of a stage-discharge relationship once in a while using more others techniques you mentioned.
With low discharges you could try using a bucket of known volume and a stopwatch.Following
- Climate change agreement in the EU. Who's the winner?
The leaders of the EU agreed on targets for protecting the climate change despite deep divisions among member states over how to produce energy. What are your feelings about the outcome of the summit?
Feel also free to answer whether it's wise to take such a young kid to such a big event (picture in NY Times)?Following
- What is the relation between p53 and mdm?
Please explain and provide some good reference regarding the relation between p53 and mdm.Following
- How do you cope with fossilized errors and help EFL/ESL students improve their accuracy?
As EFL/ESL teachers, how do you deal with such errors? As you know that fossilized errors are mistakes that language learners know are wrong but keep making.
Mohammad: Fossilised errors in language learning can be defined as routine (vocal) behaviour. If and when this behaviour impairs communication, correcting these errors means having the learners adopt a different set of fossilised habits, those described as “targets” or “correct” forms in FL/SL teaching materials.
This can be difficult for pronunciation, as Sarah notes, and I agree with Bill that raising physical awareness of both the mistake and the target is the way to go, though I doubt it that writing can play a useful role in this: speech and print are quite different linguistic modes.
If your focus, like mine, is pronunciation, these resources may help:
‘Vocal versatility and vocal fossilisation’
‘Multilingual accents’ (blog post):
‘Multilingual accents’ (journal article):
Lastly, the preference for printed forms of language in FL/SL teaching materials and the consequent failure of these materials to address linguistic prosody certainly is to blame, in my view, for “fossilised” spoken outcomes. Have a look here, ‘Rhythm clues and glues’, particularly at Carolyn Graham’s “jazz chants” rationale and methodology:
- What are the most significant determinants to the electoral/voting behaviour at the local level?
Local elections are connected with a few specific features in comparison with parliamentary elections. What determinants could we use in order to compare electoral/voting behaviour at the local level of some Post-Communist countries? Any coherent model?
Dear Miguel, Raffaele, and Yves,
Thanks a lot for your additional comments and recommendations. I consider them interesting and helpful in terms of my research activities.
Best regards, DanielFollowing
- What are the techniques that can be used to analyse a Semantic Differential Scale type data.
This scale is normally used in AHP (analytical hierarchy process) surveys. But I want to do a slightly different survey to compare just one factor and it's relationship with all other factors where as AHP consider all factors with each other.
I am not sure what you mean when you say one factor with all others. It is a mean test, correlation test or factor test?
With data collected, maybe a k-mean can separate them in similar groups.
Can you describe in more details the problem?Following
- Is it possible to perform a contrast/estimate between the same fixed-effect value?
I am interested to use contrast and estimate statements between the same fixed-effect values in my mixed model. I know there won't be statistical difference and that the estimate will be zero (0). However, I am interested in the confidence interval estimates. I would like to perform linear contrast/estimate between two groups (i.e., -A +A +B -B, or in other words, -1 +1 +1 -1). Is that possible? How do I do that using SAS (PROC MIXED)?
Though there won't be statistical difference in the effect, your interest may be realized in SAS by using PROC MIXED as if there will be. If there won't be, the confidence interval estimate includes Zero. If there will be, CI does not. The question is that you do not tell me, what is be in the second level or other levels.Following
- In the layout optimization of an EO Modulator, is the effective area to be optimized related to the optical field or to the applied electric field ?
I have to design the layout of an electro-optic modulator using a slot waveguide structure. In the papers from Christian Koos (Karlsruhe Institute of Technology), in particular Optics Express 15, N 10, p 5976 (2007), the modal effective area Aeff is the target of the optimization in order to increase the nonlinear effects. In the case of a slot waveguide, the Aeff within the nonlinear slot must be minimized. I'm wondering if this Aeff is related to the travelling optical field or to the applied electric field at the electrodes.
i really thank you for your precious explanation.
Very best regardsFollowing