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Following release of Pfizer mRNA trial data, ordered by a court to be released 75 years ahead of the original plan, we see to 31 March 2021 that 38 participants were Dead, average age 65 years. The injection history of the dead was as shown:
1 x Placebo 2
2 x Placebo 15
1 x Pfizer mRNA 2
2 x Pfizer mRNA 17
2 x Placebo plus 1 x Pfizer mRNA 2
In addition 193 people were "Lost to Follow-up" after multiple attempts to contact them with failure to respond to a certified letter sent to their last known address.
Their injection history was:
1 x Placebo 49
2 x Placebo 51
1 x Pfizer mRNA 45
2 x Pfizer mRNA 48
Has anyone found further publications following the fatality rate among the 44,820 trial participants?
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A searchable database of Pfizer mRNA trial data is available
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I was planning to evaluate the protein expression profile of a gene of my interest, in breast cancer patients. Does anyone know if such dataset ( like we use TCGA datasets to examine mRNA expression )exists?
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​Related data can be mined with the TCGA database to form mutual validation. I know of a database named CPTAC that can get results. This is the website: https://cptac-data-portal.georgetown.edu/datasets
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I don't have a cDNA kit right now. Further, if there is any protocol to synthesize cDNA from mRNA then which equipments will use. I have Dry bath and refrigerated ultracentrifuge.
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Yes, but you need to have all the required chemicals along with your template.
1. M-MuLV Reverse Transcriptase (NEB: M0253S)
2. oligo(dT) or random hexamer mixture or specific primers
3. dNTPs
you can maintain the required incubation at 42 degrees for a 30mins to an hour and enzyme inactivation at 70 degrees for 5-10min using the dry bath.
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Dear All,
I would like to ask how I should visualize the mRNA expression of a single gene (HNF1A) on a graph. I have my positive control line (HepG2), in which the expression of the gene of interest is definitely occurring, and three different hiPSC-ECs lines (one control and two mutants) in which I would like to check the expression of HNF1A (between hiPCS-EC lines; as well as hiPSC-EC lines and HepG2).
I noticed that the Ct of my reference gene (EF2) is different in hiPSC-ECs and HepG2, so I cannot use 2^(delta delta Ct). Can I just show the differences in the Ct of my gene of interest and use such values for statistics?
Thank you in advance,
Dawid
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Dawid, is this reported to work best as housekeeping gene? I would suggest using some robust ribosomal gene if possible.
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Hi Erudite scholars,
I have a quick question here. I need a Cfd knockout mice (Cfd-/-) for my experiment. So, I bred a Cfdfl/fl mice with a Cmv-Cre mice. Interestingly, after genotyping the pups, there were some mice with Cfd fl/fl, Cmv-Cre+ genotype (which can be regarded as Knockout), while others were Cfdfl/fl; Cmv-Cre- (which can be regarded as Wildtype). However, there was no significant change in the Cfd mRNA expression after conducting a a RTqPCR assay. To me, it indicate that there may an issue with the Cmv-Cre excision potential to delete the Cfd DNA flox and inactivate the gene. Any experience or suggestion on how to resolve this issue, and generate a significant Cfd Knockout mice?
Thanks in anticipation.
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Better talk to someone about your breeding scheme. Your cross would create F1 mice that are heterozygous for the Cfd floxP and half hemizygous for Cre.
You need F2 to get homozygous (if it's not lethal).
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Dear colleagues,
I will be synthesising single-stranded linear and circular mRNA by an in vitro transcription reaction (T7 HiScribe IVT synthesis Kit, NEB) followed by DNAse 1 treatment and mRNA purification. This mRNA will be lipofected into mammalian cells for protein expression. What would be the best way to preserve this mRNA for mammalian cell work? What would be the optimal vehicle for freezing? (Nuclease-free water/TE buffer/DPBS?)
At what temperature should mRNA be stored? Would liquid N2 work or -80C will be sufficient?
How thawing would affect this mRNA? How much of single stranded mRNA will be degraded upon thawing and is there a thawing routine for preserving single-stranded mRNA?
Thank you!
Kind regards,
Maria
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I agree with John Hardy Lockhart that -80 is a good choice for long-term storage. I would also suggest portioning out your mRNA into multiple tubes to avoid multiple cycles of freeze/thaw. Also, it means if something happens to 1 tube (contamination, being dropped) you will have backup tubes.
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how long does mRNA take to be translated into functional protein??
coz I'm thinking the timepoint of sacrificing the mice after treatment is quite important for Western blot analysis
thx for answering!!!!
Louis
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Very much a "how long is a piece of string" question, since the answer will always be "it depends".
Canonical maximum rates in eukaryotes appear to be around 6-9 aa per second https://en.wikipedia.org/wiki/Translation_(biology)
but this assumes translation commences immediately, there is no ribosomal pausing due to secondary structure, there are no regulatory motifs in the 5' UTR that might influence speed of ribosome recruitment, and there are no regulatory motifs in the 3' UTR that might target the RNA for degradation or sequestration somewhere non-translation-friendly.
Then, since you specific "functional protein", you need to factor in folding and post-translational modification, which for a membrane protein could be a considerable additional time lag (dystroglycan, for example, is about 60% sugars by mass, all of which need to be added before it reaches the membrane surface).
So yeah: depending on your conditions and your GOI, you could get meaningful responses within minutes, or it could be hours.
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I want to ask why sometimes the mRNA expression levels are not equal to protein levels. Thanks
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Look also a possibility of protein degradation by proteasome
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I'm carrying out a gene expression study (mRNA seq) of a gene, I noticed the BLAST results of the gene from species with same family had higher variations, but on aligning to the reference genome of the family species the variation was very less. Can I use the whole genome region that aligned with my gene of interest to comparative? rather than using the BLAST search gene sequences that had higher variations.
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In some species coding part (exons) are small part from whole gene (long introns). I am afraid that without introns you can not find the gene in whole genome. Program algorithms are adjusted to find the most longer and exact alignment between sequences. If you have short exons and long introns, there is not a way to found position of this gene in genome. You can tray to find one by one exons. Good luck !!! :-)
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We are working on biological system in which for most of the genes we do not have mRNA sequences available. We used one of the available genes for silencing experiment and want to confirm silencing levels by RT-PCR. What should be the alternative for internal control/housekeeping genes when no control gene sequence is available to use?
Can we quantify mRNA and use equal amount for cDNA synthesis as well as RT-PCR?
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Yes, you can carefully control the amount of RNA. I would combine this with a (synthetic) spike-in RNA to control for RT efficiency.
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Hello,
I am currently having difficulty in expressing or detecting the expression of iNOS. I tested its induction on different cell types: HUVEC, HAEC, CML, and with different protocols: LPS with different concentrations and serotypes, with or without IL1beta, with different incubation times: from 3h to 48h but unfortunately I did not detect its mRNA in RT-qPCR, nor its proteins at 130 kDa.
Do you have any ideas for solutions to my problem?
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Hi, could you fix it?
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This question points to a possible on-going iatrogenic public health catastrophe. Is the blood supply at risk? Clearly, further research is urgently needed. The full paper (Benzi et al 2022) is available (open access) from the publisher (IJVTPR) cited as follows:
Benzi Cipelli, R., Giovannini, F., & Pisano, G. (2022). Dark -Field Microscopic Analysis on the Blood of 1,006 Symptomatic Persons After Anti-COVID mRNA Injections from Pfizer/BioNtech or Moderna. International Journal of Vaccine Theory, Practice, and Research, 2(2), 385–444. https://doi.org/10.56098/ijvtpr.v2i2.47
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Sounds like they have an evaporation problem on their slides.
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Hello
Do you know if it is possible to profile mRNA and small RNA modifications (e.g. m6A) in human blood?
Thanks
Iddo
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Thanks Matthias,
I guess I did not explain myself very well: what i wanted to know is whether RNA modifications can be technically detected in cell free RNA from blood, which has a very low input.
Iddo
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Dear colleagues,
Could you please suggest the best way to thaw and freeze purified mRNA in water.
I have batches of unencapsulated mRNA in nuclease-free water supernatant from nanoparticles and store it at -80C. I was thawing this mRNA supernatant for a few minutes on ice, but it took too long, so I then thawed it in my hand as recommendsd by lab colleagues. Upon quantification, I found that in 2/3 samples the concentration of mRNA that is way too high and can't be real. In 1/3 samples the concentration of unencapsulated mRNA was 0 ng/uL, which is great (all mRNA encapsulated into nanoparticles), but suspicious.
I suspect that when I was thawing the supernatants in my hand, the mRNA broke apart into smaller 4-6 fragments which made reading way higher.
It shouldn't be a problem, I thought. But it seems that quick thawing breaks mRNA apart.
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I can't comment on your encapsulation or on the possible release of mRNA from the nanoparticles. But I was curious about the effect of freeze-thaw cycles on RNA integrity once, so I took samples of high-quality RNA (RIN 9) and freeze-thawed them repeatedly by transferring the tubes between my gloved hand and dry ice. After 5 freeze-thaws there was no difference and after 20 freeze-thaws I think it lost about 0.5 RIN. There's no way thawing your mRNA did that much damage to it, there must be some other explanation for the odd results you're getting. Perhaps the lipid nanoparticles do something weird when freeze-thawed? Do they aggregate during freezing?
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I would like to assess transcriptome upregulation or down regulation in my plants between treated and control. I know there is the TruSeq RNA Sample Prep Kit v2 - Set A for easy mRNA isolation and library preparation.
1. Is there a similar kit at a lower cost?
2. Alternatively l could isolate tRNA and make single stranded cDNA but this is where l get stuck. Can l sequence single complementary DNA to assess the transcriptome? If not how can l go about making a double stranded cDNA for sequencing in a Miseq platform?
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Thank you Yoav Lubelsky
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We would like to know if mRNAs from our biological fluid sample can be translated in recipient cell. The catch is that we don't know for sure what mRNA we are looking for and it would be great to label all exogenous mRNA that then enter the cell.....
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In my opinion that can be done by doing a comparative study of the gene profile by marking the EXOGENOUS mRNA before and after the incorporation of the exogenous mRNAs.
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Hi I have a problem to get the mRNA from larva of hookworm. Can anyone suggest me with the procedure?
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What have you tried so far and what was the result?
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I want to transcribe a mRNA with 120A polyA tail, and for this I need a plasmid with a site of 120 adenines. However, the company I made the quotation said that is not possible for them. I'm looking for other ways to insert a polyA tail site into the plasmid and confirm the insertion. Would anyone know how to do this?
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120nt polyA is not quite stable, you could split into 60 by 60 and place some nucleotide between. This could be synthesised as a long oligo, annealed and sub-cloned into a vector by digestion ligation. Here is the reference
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I found in my scRNA-seq data that H4C8, whose mRNA lack poly-A tail, was present in some cells.
why such mRNA can be captured after mRNA enrichment by poly-T?
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I think they firstly flag the mRNA before sequencing with poly-A tail. After that they can be seprated by enriching with Poly-T.
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Any direct or indirect protocols that can be used to clone.
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Yes and it is done all the time to get rid of the introns in most cases. You reverse transcribe your RNA then use that as an input for PCR.
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Hi
VMD tkconsole either gives psf pdb of my structure without adding hydrogen bonds and fixing other things or using autopsf plugin causes crashes. All I feel it maybe happening because of lack of topolgy file of m7G attached at the tip of mRNA chain.
I have used following commands in tk:
%topo guessimpropers
%topo guessangles
%topo guessdihedrals
%topo guessbonds
%mol reanalyze top
%set all [atomselect top all]
%$all writepsf mymol.psf
%$all writepdb mymol.pdb
Please help me in generating psf pdb of whole complex.
Thanks
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I want to purchase a pre designed siRNA to silence my gene. So, by searching websites of companies such as Thermofisher, I have found a list of 10 siRNAs for my gene of interest. They differ in the location on the mRNA that they are targeting, given that my target gene consists of only one exon (there are no introns). So, what are the criteria based on which I can choose the best siRNA for my gene.
In addition, what are the best biotechnology companies that provide RNA interference products? I found a lot of papers using products from GenePharma, a company based in China. So, for those who tried RNA interference products from this company, what is the quality and efficiency of silencing of its siRNA products?
Thanks in advance
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I know that Sigma-Aldrich and Thermo-Scientific have it. You could try them
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Text book always said that most important job of RNA is protein synthesis - assembling amino acids into proteins. Messenger RNA carries instructions from nucleus to ribosomes. Transfer RNA transfers each amino acid to the ribosome as needed by the code of the mRNA molecule.
However, it seems that it never mentioned the metals in the protein.
May I miss some knowledge in this field?
Where are the metals?
How the RNA to decide the metal center in the protein?
Any hints?
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Thanks for your reply.
I am not sure if my question is clear. What I need to know is the factors that contribute to the selectivity of the metal center in metalloenzymes and the role, if any, the corresponding RNA/DNA plays in this process.
In other words, is the metal center first, then the amino acids -controlled by RNA to fit its structure to form the metalloenzymes, or if the amino acids -controlled by RNA first and then select the metals to form a stabilizer protein?
If it was the second case, the metal availability in the cell is important, which is always linked to the environment. However, the metal center seems quite stable and it does not change for a metalloenzyme.
I prefer the first case. However, I do not have any references to support my idea. Any hints are welcome
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The recent announcement that a popular culture icon suffered facial paralysis from Ramsay Hunt Syndrome (RHS) has led to speculation that it could be associated with COVID-9 Vaccination.
The first report of the association of RHS with Pfizer mRNA vaccine was published by researchers in Hong Kong in 2021.
2 days after his first dose of Pfizer a young man had fever and pain in his right ear. Investigators found Vesicles in his right ear and canal, and he subsequently suffered Vertigo, Tinnitus and Loss of Hearing, Facial Palsy, Tongue Numbness and Dysgeusia.
Thousands of cases of Herpes have been lodged with COVID-19 Adverse Reaction notification schemes in many countries and it has been reported after receipt of Adenovirus vector vaccines as well as those delivering mRNA.
There are now many reviews of cases of Herpes after the jabs.
Experts in the field suggest that Varicella Zoster Virus-specific CD8 cells may be temporarily incapable of controlling the Herpes after the massive shift of naive CD8+ cells in response to the inflammatory impact of the vaccines.
When investigating the full range of symptoms of each individual report, perhaps Herpes has not been adequately tested for in the jab > Herpes activation > Multiple Organ Damage Cascade?
Are other dormant viruses activated by vaccine-related immunomodulation, e.g. Hepatitis?
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A friend kindly searched the TGA Database of Adverse Notifications in Australia which is reporting 11 cases for the specific search term Herpes Zoster Oticus (also called Ramsay Hunt Syndrome) for Pfizer COVID-19 vaccine (Medicine suspected).
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I invitro transcribed the DNA template of me mRNA vaccine using mmessage mmachine ambion invitro transcription kit. The nanodrop results are good and if ound the mRNA to be 60 ug in 50 ml volume of NF water. when i tried to visualize the mRNA vaccine on RNA denaturing gel I was unable to detect any band, neither untailed nor tailed. Although the bands of control mRNA are visible as both untailed and tailed form. This was my 3rd trial and im unable to identify the main issue behnd although i follow the protocol carefully every time. Kindly share your useful tips.
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Thank you for the question and congratulations for the very good nanodrop results.
For the visualization of mRNA vaccine on RNA denaturing gel, my suggestion is to check the PCR cycling parameters, taking into consideration of the length of the control and your sample. Somewhere using too high of an annealing temperature can lead to low product yield.
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how i isolate the newly synthesized mRNA only?
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I'm sure someone will suggest an easier method for accomplishing this task but, I wonder if you could take advantage of click chemistry to capture only newly synthesized mRNA.
I imagine there are uracil analogs that possess "clickable" chemical groups (i.e. azide group), which you could use for capture. For experimental setup, you could grow your organism, treat it with the clickable uracil, allow time for new mRNAs to be synthesized, extract RNA, purify mRNAs containing clickable uracils, then perform your analysis using qRT-PCR.
This is certainly not a quick solution to your problem - hopefully someone can suggest something easier than this!
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The Bioanalyzer basically looks at the quality of rRNAs and generates the RIN value.. Is there a way to access the quality of mRNA or even pre-RNA?
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The BioAnalyzer RNA 6000 Pico chip type can be run in mRNA mode. This is done by choosing 'eukaryote mRNA pico' as the chip type in the instrument software when setting up the run. When this is selected, the BioAnalyzer will report the mRNA quality by describing the percentage of rRNA contamination, instead of giving a RIN. (RIN is calculated by the ratio of 18S to 28S rRNA, and so when rRNA has been selectively degraded or removed, RIN is meaningless anyway.)
Percentage rRNA contamination is very useful when you want to determine how effective your mRNA isolation or rRNA depletion was, and whether it's comparable between samples. It doesn't tell you very much about whether your mRNA is degraded. The best way to do this on the bioanalyzer is by eye - you can find images online of good quality mRNA profiles on the bioanalyzer and visually compare yours to these. Any size-shift towards the left (smaller average fragment size) indicates some degree of mRNA degradation. Degraded material will also show up as a peak between ~25 and 250nt, so if some samples have much more signal in this region, they are likely more degraded. You should also ALWAYS take an aliquot of your sample before mRNA enrichment and run this on the BioAnalyzer as well, as knowing the integrity of your starting material is very important (and it needs to be consistent between samples, or at least similarly distributed and similarly variant between experimental groups that will be compared.)
There are also some differences between ribodepleted and poly-A selected mRNA profiles on the BioAnalyzer. Assuming good quality starting RNA (RIN 8-9 or higher), poly-A selected mRNA should have strong signal within the mRNA size range (approx 1000-4000nt) but very little signal in the small RNA region (~25-250). Very few transcripts this short are polyadenylated, so if you're seeing signal here, it means you've captured 3' fragments of larger mRNA species which are degraded. Conversely, ribodepletion selectively removes ribosomal RNA while leaving native small RNA in the sample. If the sample quality is high, then you will see a similar profile in the small RNA region of ribodepleted samples as you saw in the input material. Many distinct peaks in the small RNA region is indicative of native small RNA (high sample quality); a single big peak is degraded material. This assumes your samples were cleaned up on a column: any bead cleanup will remove the native small RNA fraction from the sample, as well as most of the degraded material. If a bead cleanup has been performed, you will need to estimate the RNA quality by looking at the intensity and size distribution of the stuff in the mRNA size range, and disregard the small and degraded RNA region, which will be missing.
It is definitely also worth checking RNA integrity by qPCR if your experiment is important and if you can spare an aliquot of sample to do this.
Sorry this is long - advanced bioanalyzer interpretation is complicated - happy to clarify if you have any more questions :)
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We have received 2ml pleural fluid. We have extract RNA for mRNA library preparation. Please suggest which method or kit needs to use for extarction.
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You can use for example Plasma/Serum Circulating and Exosomal RNA Purification Maxi Kit (Slurry Format, Norgen Biotek, Canada, cat. no.
50900). We used it here in the study of ascites:
Ensure, what type of content you are isolating, either the whole pleural fluid fraction or cell-free supernatant fraction. You can also separate the cells. In case of cells isolation, you can use TriReagent/Trizol, Mirvana, etc. For fluids, alternative isolation kits are also available, for cell-free fractions you can use those for plasma RNA isolation. Always ensure that kit isolates the fraction of small RNAs.
Goog luck.
Luděk
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I have generated a CRISPR/Cas9 knock-out zebrafish of my desired gene (referred to abc-/-) and an overexpression line of abc by using Tol2 transposon (referred to Tg(hsp7:abc)). All the phenotype characterisation and mechanism studies were done. However, one of the reviewers suggested that the loss of function of the abc-/- should be restored by overexpression of Tg(hsp7:abc). However, crossing the two lines together demand another 6 months and sampling injecting mRNA of abc to embryos is not suitable since we need to observe the behaviour phenotype at 7dpf and the injected mRNA will decay at that time.
Although I agree with the reviewer that the rescue experiment is necessary. But I think the overexpression lines have already shown the opposite phenotypes and not all the studies perform the rescue experiment. So I want to know if there are some other ways to respond to the reviewer.
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Zexu Chen In this case, politely reply to the reviewer that the rescue experiment will take 6 months. Mention why you think your data is valid even without the rescue experiment, just like you did here, and in addition, explain if this experiment is the main experiment of the article or not. If not, mention it. Then, revise your text to use less harsh language, i.e. write in Discussion that "one of the potential drawbacks of our study is that no rescue experiment was performed". Then (in the answer to the reviewer) write "we have modified our manuscript to highlight the fact that the rescue experiment was not performed. Write then, "However, there are several papers where it was not performed (cite said papers), and conclude to the reviewer that "due to the time constraints and budget we ask the reviewer to consider modification of the article's text as an appropriate response to this problem".
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I have run mRNA on 1.5% TAE gel but the band is ~4kb above its actual size. I have heat my sample at 90oC for 2 mins to avoid secondary structures. But I haven't seen the mRNA on actual size.
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Yes, I have restricted plasmid with XbaI (Neb) at the end of the gene. This enzymes prodces 3' sticky ends.
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I am preparing to design primers to clone a Leu2 maker from a plasmid into Saccharomyces cerevisiae (BY4742) in place of a native gene via homologous recombination. However, I struggling to get my head around the genetic workings of the gene orientations of both the Leu2 gene (from plasmid PAG305GPD-ccdB) and Htz1 from (BY4742). From what I can see, both the Leu2 and Htz1 are coded for on their respective negative strands.
As I understand it, transcription always takes place 5'-3 'direction against the antisense strand, which in this case would create mRNA strands with this kind of sequence 5- XXXXXXXXGUA-3'.
My confusion is this - How is it then translated into a protein if the 'AUG' recognition codon is in reverse. Can a ribosome move from the 3'-5 'end of a strand or is it rather that in this case transcription takes place against the sense strand? Or am I misunderstood something fundamental along the way?
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Fundamental misunderstanding, I'm afraid. Though it's great that you're thinking these things rather than just assuming you know what's going on.
DNA is always transcribed in the same direction, it's just that DNA has two strands, which run in opposite directions.
There is no difference between a gene going 5'-3' on one strand, and a gene going 5'-3' on the other strand, even though those two strands are wound together such that each runs in the opposite direction to the other.
A gene can be encoded on either strand of the DNA, as here. The RNA polymerase copies from one strand, and the other strand in that region is ignored. RNA polymerases only 'see' one strand at a time, and that's the strand they bind to
(slightly confusingly, they bind to the non-coding strand, and they travel in a 3'-5' direction, because they are producing a complementary sequence, i.e. a copy of the coding strand, which consequently runs 5'-3').
The resultant mRNA is single stranded, is a copy of the coding strand sequence (with U in place of T, obviously) and runs 5'-3'.
Note that genes rarely overlap between strands (i.e., HTZ doesn't overlap with PDB3) because this would require a region of shared anti-parallel coding sequence (which is both much more vulnerable to mutational change, and a lot more challenging to evolve). Some viruses do in fact have multiple reading frames that overlap on antiparallel strands, because viruses are awesome, but this is because viruses are under huge selective pressure to have small genomes, while higher eukaryotes have essentially zero pressure for this (the human genome is like 90% filler sequence, for example).
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Hi everyone, im interested into knowing the quantity of spliced and unspliced mRNA for a paticular gene. I thought using primers for mature mRNA (exon to exon) and other set of primers to quantify primary transcript (pre-mRNA) using a part of intron and its contiguous exon to amplify unspliced mRNA. Is this procedure correct? Can it be donde by RT-qPCR if i treat my extracted RNA with DNAsa (so theres no genomic contamination)?
Thanks
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Not with the standard cDNA synthesis protocol. It relies on the dTTT primer to bind and amplify mRNA to cDNA based on the mature mRNA with a polyA tail.
Introns are removed and exons are spliced together BEFORE the polyA tail is added.
But there are protocols for you. Do a literature search.
Good luck!
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Hi,
I have a question regarding of ddCT method of qPCR.
Various threads have pointed out that all statistics should be done with dCT or ddCT because dCT follows a normal distribution.
I understand that dCT in single cells follows a normal distribution, since gene expression in single cells often follows a log-normal distribution, as shown in various papers (i.e, Bengtsson et al. Genome Research (2005)).
However, if mRNA is extracted from a large number of cells (e.g., more than 1.0x10^6cells), then the mRNA solution is the sum of mRNA extracted from a cell population whose gene expression follows a log-normal distribution, what happens in this case?
Does this mean that the amount of mRNA of interest between samples follows a normal distribution, according to the central limit theorem?
In this case, can dCT follow a normal distribution?
I would appreciate your response.
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Dr. Wilhelm,
Thanks for your quick reply.
I am not familiar with math or statistics, so your comments are very helpful. Thank you very much for your valuable information.
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Hello everyone,
I want to measure the expression change of some mRNA, miRNA, and LncRNA at once under a specific condition.
Since I am going to do relative quantification, is there any RNA extraction reagent that can extract all RNA molecules simultaneously?
I am willing to hear if you have other solutions rather than a reagent for all molecules.
Thanks for your help.
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if you have a budget go for small RNA sequencing and Transcriptome analysis.
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Hi everyone,
I am trying to do an MD simulation of a peptide(with synthetic amino acid and aliphatic linker) and mRNA using GROMACS. OPLS and amber14sb_OL15 force fields are selected to model peptide and mRNA respectively. When I perform the ion addition step (group command), I received the following error.
Can anyone suggest to me how to solve this issue?
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Thank you Mohamed Khedawy !
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Hi everyone, I'd like to analyse mRNA expression level of gene in a treatment to wild type and analyse mRNA expression level gene between treatment in all group treatments.
I used T-test and there is no data significantly appeared. Then I tried F-test and the data become significant. Is anyone experience this? Thankyou
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To get a reasonable answer, I think you'd need to clarify the question.
To me, it sounds like you have, say, 5 groups: say, 4 treatment groups and 1 wild type. You then used a t-test to compare 4 vs. 1 ? And then an analysis of variance to compare among the 4 treatments ?
Also, note that t tests and F tests can be used for all kinds of totally different purposes. Just saying "t test" isn't clear what you are doing at all. If I can try an analogy, it's like saying "I used a metal tool and a wooden tool". And we're like, "Used them for what? As a hammer? As a wrench? As a screwdriver?"
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Hi, does anyone know of any post-transcriptional mechanisms that could explain no change of the transcript (mRNA) but still impact the protein?
so modifications that may show the protein's expression low in mRNA but the protein is still expressed in high amounts after translation?
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Malcolm Nobre thank you this helps
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For a triplicate, I obtained Cq values of: 39.03, 0, and 38.99. How would I average these? There's very low (if any) expression of the mRNA. Would I need to repeat these?
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The 0 would very clearly need to be discarded. You can then average the other two values together, but I agree with you this value is high to the point that I'm not sure I would believe you are getting actual amplification of a product here. What do your melting curves look like? Did you save the the reactions after they were done being run? If so, you could run them on a gel and see if there is an amplicon at the predicted size.
Usually I have a set value, above which I consider it "not detected;" this value is going to be specific to your instrument and set up though, and may need to be optimized.
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Hi all,
I'm looking for COVID mRNA vaccine materials for mice experiments, something just like pfizer/moderna. We are investigating the potency of our vaccine candidate working as a booster for these popular mRNA vaccine, but it seems impossible to get our hands on some mRNA vaccine materials. Where can we buy some of those for mice studies? Or do we have to initiate collaborations with someone to obtain those materials?
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Thank you for answering this question! I'm located in Boston.
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I want to align mRNA with the genomic sequence. I was using MGAlignit earlier but the server is not responding now. Do you know of any alternate tool?
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Try out my own developed gui based allignment tool, BLGAST-By-SKUAST on sourceforge
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Some research papers on mRNA based vaccines have provided their mRNA Vaccine construct with T not replaced with U (making it a DNA sequence rather than RNA), while some papers have given their sequence as in RNA. I can not understand the contradiction, if someone can help me with this.
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Sequence of the vaccine constructed according need of protected vaccine irrespective to kind of sequence either U or T
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Secondary structure prediction
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what do you mean that your protein does not have an alpha elix? Do you mean any alpha elix? or that it miss a trasmembrane helix that could be necessary to anchor your protein outside the cell surface?
Because the are proteins in nature that has B-barrel folding (eg. bacteiral outer membrane proteins). But of course it depends from the specific nature of your protein. I do not think that with out know more information about your proteim we can provide you any reilable tips.
best regards
Manuele
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Hello,
I have been struggling with the electroporation of PBMC with HIV-I mRNA. Although the quality of the RNA generated is good, and the handling of the cells in meticulous, the frequency of successful electroporations is very low.
Since I work on HIV-I infected patients, their consequent variability is of Course taken under consideration. However, the rate of successful electroporations is still too low to what is expected.
Are there any tips or advice regarding viral mRNA electroporation I can get? It´ll be much helpful and appreciated.
Thanks in Advance!
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hi Boutaina El Kenz,
have you solve your PBMC electroporate issue? recently I struggling with that ,,,,,could you kindly give any guidence for me ? @Boutaina El Kenz many thanks and appreciate.
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Hi,
Has anyone tried commercial kits for polyA tailing of in vitro transcribed mRNA (e.g. NEB E.coli polymerase kit)? I keep getting inconsistent results, some mRNAs were tailed but some are not, despite the reactions being set up in parallel. Is this a common problem for polyA tailing using E. coli polymerase? thanks!
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Are your samples all the same quality before you start the tailing protocol?
I cant see no reason for the protocol to work only with some samples other than the samples have different degrees of contamnation
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Starting from the genetic sequence of an mRNA, is it possible to allow the self-assembly of graphene nanoparticles (GQD - Graphene Quantum Dots), in such a way as to obtain certain circuits, with the formation of specific electronic components at the nanoscale? With what correlation between the triplets and the electronic scheme?
Thank you.
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Kira, Unfortunately, this question is not in the area of my expertise. I would be conjecturing too much to qualify the answer as scientifically validated. I apologize.
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I need to find a Mammalian cell line that can translate mRNA of a bacterial gene
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Any mammalian cell will translate an mRNA provided it has a good ATG (Methionine) translational start site, usually. A/GXCATGG/A, also known as a Kozak Sequence. What is important is a eukaryotic transcriptional promoter to express the mRNA in mammalian cells. Once you make the mRNA, the ribosome does not know whether the coding sequences is bacterial or not. So any mammalian cell can translate a bacterial gene provided you construct the expression plasmid appropriately.
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good morning everyone, I would like to synthesis some mRNA fragments but i don't have too much experience in this fild, I know I can carry on on two different ways:
- PCR transcription
- DNA plasmid coding the mRNA fragment
can you give me some advise? wich one are the best option?
thanks a lot
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thanks a lot for your help. I don't have jet the mRNA fragment so I have to decide in way to produce it;
can you tell me the benefit and th elimits to use PCR and the Plasmid? like time, cost, difficulty of the procedur ect ect
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Why is Poly A present on certain RNAs like mRNA and several non coding RNA and not in other RNAs like rRNA and tRNA?
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Hi
The 'PolyA' tails are not encoded in the gene and added later by PolyA Polymerase. The genes that contain a PolyA signal and a PolyA site generates transcripts which can be polyadenylated, however there may be additional factors involved in the process. It may be possible to insert such sequences in genes lacking natural PolyA tails. Unlike mentioned by @Loius above, the rRNA and tRNA are noncoding RNAs ane do not code for any protein. The presence of 'AAA' codon and relation to Poly A tail is not clear.
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Hello everyone
I am working on next aim of the project which is to produce mRNA vaccines in plants mitigating the needs to purify them and can be consumed as a part of food. I am planning to target my DNA construct encoding the S gene of Covid19 to the chloroplast genome as they have their own transcription machinery and lack gene silencing. I have few doubts in this regards:
a) I am planning to clone my S gene into deconstructed viral vector construct? shall I perform biolistic method to target my transgene into chloroplast genome? I want stable integration into the chloroplast genome (or is their any other alternative)
b) I will do genotyping to confirm the transgene insertion
c) How I am gonna make sure that S gene are not getting translated and remaining as mRNA for mRNA based vaccine? I can do northern blot but since mRNA gets translated/degraded, how the genetic design will make sure that enough mRNA will be their for therapeutic effect?
d) Once everything is confirmed then offcourse we are going to test in mouse model
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Hello everyone,
I am going to perform a pull-down assay on a specific mRNA that I have in mind. I was thinking to use streptavidin magnetic beads. Would anyone please be able to share the protocol? I prefer using a kit but any suggestions/related articles would be appreciated. Thanks.
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I have commercial batches of cell free extracts which I use to do protein expression by just adding a plasmid to it. I can quantify the amount of protein produced over time (translation product) using mass spec. How can I quantify the amount of mRNA produced over time (transcription product)? The sample will be a complex mixture of loads of proteins and other kinds of RNA (tRNA, rRNA). Any way to precipitate all that and just keep the mRNA content and run a urea gel with it? Or some other technique?
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You can use polyT column to separate mRNA which should have a polyA tail.
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Can anybody brief me on the zeta potential of mRNA?
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do you have any DLS data file you can show? The quality of the DLS data and repeatability will determine whether the hydrodynamic size of your sample is reliable. If you do not have the raw data, take a look at the z-average and PDI, and compare that to the size you expect. It may provide a first pass at how reasonable the diameters are.
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Hi,
Is there any database to identifies which protein isoform is expressed in a particular tissue?
I want to design primers for real-time PCR on cartilage samples. I need to be sure which mRNA variants are specific for cartilage.
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Hmm, hard to say...
It seems that isoform uc002bna.2 (http://genome.ucsc.edu/cgi-bin/hgGene?db=hg19&hgg_gene=uc002bna.2&org=human) is expressed in most of the tissue types (see the image in the attachment) ... but there are no data for cartilage tissue type specifically
So you may risk it and proceed further with primer design, or you can try to find expression of your isoform in some cartilage-specific public RNA-seq data (laborious)
Martin
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Hello!
I isolated some T-cells with some very interesting TCRs from primary cultures. I sent them to Genewiz to get chromium single cell TCR sequencing done, however the sample viability was super low. I sent them 8 more vials so that they could do a dead cell removal and then isolate the live population and perform single cell sequencing on the remaining cells. The sequencing results show that whatever is left over after DCR is most likely another contaminant cell type, not a TCR. I now only have one vial left, so whatever I choose to do next is very critical and essentially has to work the first try.
At this point I dont care about chain pairing, I can piece that back together afterwards by trial and error, I just want to get some data from these cells. I was wondering if anyone knows if I can thaw my cells directly into RNA later and then do either normal NGS or another single cell sequencing method to get any info on the TCR sequences? Should I just amplify the TCR regions on thawing with some kind of primer pool and then send that for NGS? In general, what's the most robust process for getting out TCR information from low viability samples?
Some other notes:
1. I didnt personally do the T cell isolation but my thinking is they were pretty much exhausted at the time of freezing which is why we have viability issues on thawing
2. They were frozen in 10% glycerol + 10% FBS in a Mr Frosty at -80C and then maintained in LN2 and shipped on dry ice.
3. Observed viability is ~30% on thawing however this could just be the contaminant cell population....
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Definitely a fair assessment, Ive made other samples that have similar properties as this, but none of the other samples Ive generated have responded with as much vigor as this sample.
Definitely will invest in generating another panel of T-cells but from what I can tell so far, this sample had a particularly rare phenotype that i may or may not see again in a relatively limited panel size. every once in a while I remember I have this last vial and wonder if I can do anything with it, in the end its always for the birds...
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I am working with primary human fibroblasts from patients with severe metabolic disease. I am interested in using RNA/mRNA re-programming of these fibroblasts in order to obtain iPSCs for downstream functional characterisation.
In recent years, numerous kits/protocols have cropped up purporting to tackle this issue. I was wondering if you could share your experience of this approach and which method you found most effective?
Many thanks,
Rob.
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Hi Jing Liu . That sounds very promising. I will definitely check it out. Thanks for your answer!
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I want to do an MD simulation of GFP (Green Fluorescence Protein ) mRNA. But it's too long containing around 800 bps, impossible to do all atomistic simulations.
May I know what are the possible ways to handle this kind of situation other than coarse-grained simulation?
Thank you in advance!
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What is the purpose of the simulations?
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Hi everybody,
I am using the mMESSAGE mMACHINE Kit (Ambion) to generate capped mRNAs in vitro, but since I also want to add a polyA tail to those transcripts, I decided to use the Poly(A) Tailing Kit  (Applied Biosystems) which is the recommended one for capped mRNAs generated with the mMessage kit.
The polyA tailing kit suggests to use 'a completed, DNase-treated mMESSAGE mMACHINE reaction (20ul) at room temperature'. My question is if that RNA needs to be previously cleaned-up from unincorporated nucleotides and enzymes or if it can just be use straight after the DNase treatment step.
I have previously used a different kit for polyadenylating which required 10ug of a cleaned-up capped RNA as input material for the polyadenylation step, and this is why I am concerned about including (or not) this extra clean-up step (I know for sure I will need to clean up after I have my polyadenylated products...my concern is if I also need to do it before).
I would appreciate any suggestion on this matter.
Thanks!
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Hi Natalia,
I recommend doing a clean-up before setting up polyA tailing reaction, as the unincorporated nucleotides may hinder the tailing reaction.
Also, I was wondering if you had any problems with the polyA tailing enzyme? I have tried NEB polyA tailing kit but kept getting inconsistent results, some mRNA were tailed while others were not, despite the reactions being set up in parallel. Any information will be appreciated.
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Hello everybody,
In my work, I'm actually interesting about mRNAs-contained in a protein complex in yeast S.cerevisiae.
We perform in the lab the immunoprecipitation of these mRNAs (RIP) from a S.cerevisiae strain for which one of protein complex is tag, and we now wanted to identify these mRNA by sequencing. The mRNA amount control is mRNA immunoprecipitate from an untagged strain.
But how can I determine the number of reads that I have to choose for the mRNA sequencing ?
Thanks !
Best regards
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Vincent Pacini , can you detect the mRNA concentration by nanodrop? It should not be a problem as long as you can detect the amount with nanodrop.
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Dear all, I have found white precipitate in my transcription buffer which is expired 6 months ago (Promega ribomax sp6 kit). I used the buffer to synthesis mRNA and run a luciferase assay. Unfortunately, I didn't get any luminescence signals.is it because of my transcription buffer or any other issues?
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The precipitate was DTT. Just be patient to dissolve it. If you don't trust the buffer, make your own batch (you can find the buffer composition in the kit's manual). Have you quantified your RNA or visualised it otherwise prior to translation reaction?
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I have been trying to check the expression of a pro-inflammatory cytokine between differentiated wild-type and knocked-down THP-1 macrophages cells. When I stimulate the cells (both at 24h and 6h)mRNA expression is reduced in kd-THP1 cells (by qPCR), but not the protein (checked by ELISA). The protein levels are similar between both the cells, when I have significant reduction in the mRNA levels. My question is, if anyone faced the same issue or any way that I can overcome this issue?
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This is not an issue at all and very common in secretome analysis. What matters for you is that both ELISA and western blot goes parallel with each other.
The explanation would be that your gene of interest is subjected to post transcriptional modifications, and that's normal and common (which is the case IL-6 for example)
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I would like to study correlation between four transcripts (fold changes of mRNA expression) at different time intervals (5 time points). How can I perform this analysis?
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Try Correlation matrix on R Programming. Try corrplot( ) package in R
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Suppose, I have a treatment group and a control group. I have measured the relative expression (delta delta ct) of two genes by real-time PCR analysis. Both the two genes are analysed from the same samples and the same reference gene was used. In this case, if one gene has higher relative expression than another gene, can I say that gene is highly expressed than the other gene. My question is if the relative mRNA expression from two genes can be compared? another question is, is it ok to produce a heatmap from relative expression data of different genes?
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Thanks a lot for your answer and suggestion professor Iman Hassan Ibrahim and Dr Abhijeet Singh
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Nucleases are proteins that break phosphodiester links in DNA and RNA. The poly-A tail is added to mRNA in eukaryotic cells to protect it from degrading in the cytoplasm and allowing it to efficiently translate and deliver the polypeptide sequence.
The question is whether the mRNA that was transcripted would decay if prokaryotic organisms possessed a nuclease. Simultaneously, the question arises: how can it be translated into a polypeptide sequence?
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mRNA stability in both prokaryotes as well eukaryotes is one of the mechanism of gene regulation . Evidence for former one are very recent . Following mechanism are responsible for protecting prokaryotic mRNA from degradation
1.5’ terminus is triphosphorylated, a situation that appears to inhibit the binding of certain endoribonucleases . In addition, prokaryote the immediate 5’ terminus is not involved in the initiation of translation, but rather the existence of a downstream ribosome binding site (RBS) promotes association with the 3’ end of the 16S rRNA. The coupling of transcription and translation in prokaryote is another factor that is unique in prokaryotes and affects mRNA stability in that failure to translate an mRNA usually results in its rapid degradation . Further The presence of multiple open reading frames (ORFs) in a single polycistronic transcript is a further feature of prokaryote transcripts that is not found in Eukaryotes
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We are working on a new parasite species and want to choose the most abundant enzyme in order to prepare vaccine against it; we thought to measure the related mRNA for each enzyme by qRT-PCR. However, we face a problem in the data analysis as we do not have a reference sample.
Is there a way to compare the mRNA expression for different transcripts in the same RNA sample without using delta delta CT method which requires having a reference sample?
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You have to make your own control. Try using essential enzymes for the life, its expression should be ubiquitous.
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In the attached image, it shows the cleaved PARP densitometry analysis from western blot. Cleaved PARP is commonly used for apoptosis. Treatments (A and B) were tested along with control and the two time points were used 8h and 24h.
I'm trying to run qPCR experiment looking at mRNA levels of apoptosis markers such as BCL2 and PUMA. My question is do I choose 8h for qPCR assuming that mRNA comes before protein so looking at an earlier timepoint is better? Or do I choose 24h since I see the significance in cleaved PARP at 24h in western blot?
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Why not both?
qPCR is pretty cheap, and you'll get more information about what's going on if you look at both 8hr and 24hr timepoints.
Ideally you'd have more timepoints, even. 0, 2, 4, 8, 24, for example.
Plus be aware that you'll only be measuring transcriptional responses: any apoptotic responses using existing, already translated BCL2 protein (for example) will not be reflected in your qPCR assay.
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I've started working in a new lab, i think there is some problem with equipment or loading dye or something.
They have been having this problem before i got here and have been ignoring it, but I'm supposed to do RNA work.
Lanes are as follows from left to right
DNA ladder 1kb
Standard ssrna 7kb
Clean IVT product 8kb (low concentration)
Clean IVT product 8kb (high concentration)
Space
6-11-mutant Mouse mrna samples
12-14 WT Mouse mrna samples
Used 1/2 TAE (Have gotten similar results with 1 TAE)
Used invitrigen gel loading buffer for all samples (not the dye i used to use but that's what they have here, recommendations for better dyes are also welcome).
The RNA is not degraded, running same RNA in different lab with different equipment looks fine.
What might be the problem what can i do about it?
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In response to Dr. David Farringdon Spencer, I completely agree with the clarification. I suppose that in this lab they didn't have appropriate solution that can help solve the problem, if this is the case, maybe the problem is something else.
Working in some laboratory very bad equipped, without any idea of how to handle RNA, I am drawn towards this "companion in misfortune"
Valeria
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I am planning a study where I want to select a few potential miRNAs that are responsible for inhibiting protein translation from a particular gene.
I am new in this area so it would be helpful for me if I get some guidance on how to select the miRNAs for a specific gene and if there is a way to determine miRNA and mRNA interactions with any bioinformatics tool before moving forward.
I have selected a few miRNAs for my gene from TargetScan, Mirdb, and Mirtarbase but I want some other opinions.
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Sameh H. Mohamed Yes, different target prediction tools uses different algorithms, so there are differences in predicted targets. So, to avoid confusion, focus on one tool, you can prefer TargetScan whose results are well-optimized & established.
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Best way for plasma separate for rna extraction (miRNA and mRNA)
incase there is no cool centrifuges
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Think about it: plasma is usually at a near-constant 37 degrees. So whatever mRNA content it will contain will...probably be stable, at least in the short term, at anything at or below 37 degrees.
Thus: extract plasma as normal, using a room temp centrifuge, and then freeze the plasma. Freeze the plasma in modest aliquots (200ul is practical), so you don't need to keep freeze/thawing it. Store at -80.
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Hi, I need to have a dataset including the expression level of a particular miRNA and a particular mRNA in tumor (digestive cancer :Colorectal+gastric)+ its matched normal tissu across samples provided by TCGA database .What exactly should I do? thanks
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Ganesan Jothimani sure, you are welcome, this is my email "fatma.1990@hotmail.fr''.
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way of preservation blood (plasma) for RNA extraction less than 24hr is there any effect ?
incase I want to keep it for 3day before extraction which is the best way for preserving
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Hi Raya
keeping safe samples in tubes during days is never good for further analysis. the best way therefore in your case is to use tubes as the PAXgene tubes from BD, but it's expansive:
all th ebest
fred
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I would like to learn if there is any tool to design mRNA? or is there anyone who know about how to design mRNA for an in silico study?
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Annemarie Honegger I sent you a private message.
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Hello! I've been studying about ADAR which stands for Adenosine Deaminase Acting on RNA. I heard that ADAR only works on double stranded RNA and changes mutant A to I. But isn't mRNA in our body single strand? I wanna know how there can be double stranded RNA(the target of ADAR) in our body.. plz help me!
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Hannah Hwang You're Welcome.
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They were expected since the publication of press releases, the detailed scientific data on the Pfizer-BioNTech vaccine, BNT162b2, have just been released in a 92-page document. It summarizes all the information concerning the clinical trials of the Covid-19 vaccine, from phase 1 to phase 3, as well as specific elements on the effectiveness according to the profile of the patients.
As a reminder, in a press release released on November 18, the American pharmaceutical company and its German partner assured that their vaccine is 95% effective without further clarification. As a 90-year-old received the first dose of this vaccine in the UK the day before this document was released, let's take a closer look at the safety and efficacy of BNT162.
BNT162 vaccine is a messenger RNA vaccine. The syringe contains multiple copies of SARS-CoV-2 protein S mRNA encapsulated in lipid microdroplets. When they enter cells, mRNA is translated into S protein in the cytoplasm. Cells express this antigen on their surface, which stimulates the immune system.
At no time does mRNA interact with the cell nucleus and DNA. It is also rapidly degraded by cellular enzymes without leaving a trace.
The Pfizer-BioNTech vaccine has a classic safety profile
Who are the participants in this clinical trial. 37,706 people volunteered, all over the age of 16 and having never had Covid-19. The average age of this workforce is 50.2 years, a relatively young group, with 42.3% over 55 years.
Over 80% of the participants are white people, mostly from the United States or Argentina. Finally, about 70% of the participants are overweight (34.9%) or obese (34.8%).
The participants are randomly divided into two equal groups: one who will receive the vaccine solution (a dose of 30 µg) and one who will receive a placebo, namely a saline solution. The protocol is the same for everyone, two intramuscular injections spaced 21 days apart.
How did they react to these injections? The inconvenience was more frequent for people in the vaccine group, which is completely normal and even reassuring. The vaccine plays its role by stimulating the immune system, which also causes some symptoms related to inflammation.
Locally, participants experienced pain and redness at the injection site. At the systemic level, fatigue, migraines, muscle pain and more rarely fever (<38.9 ° C) have been recorded. Side effects that can interfere with daily life, but disappear in a few days and do not require medical attention. Note also that these side effects are less common in people over 55, because of their less robust immune system.
In summary, the Pfizer-BioNTech vaccine causes the same minor side effects as most vaccines. Long-term data is still lacking, but at the moment there are no alarming signals.
The effectiveness of the vaccine by age group
What about the effectiveness of the vaccine? Good news, the results presented by Pfizer are good and reliable. Vaccination is 95% effective when all participants are considered. The 95% confidence interval is between 89.9 and 97.3%, which means that the BNT162 vaccine is at least 89.9% effective according to these data.
When we delve into the data by age group, we observe that the vaccine is a little less effective in people over 65 years old, "only" 94.7%. This is also due to the aging of the immune system which responds less well to vaccination. It is even lower for the “all others” group, which includes ethnic minorities such as Amerindians, Alaskan natives, Asians, and people from the Pacific, namely 89.3%. This is also the case for people vaccinated in Brazil, there, the effectiveness of the vaccine is only 87.7%.
the cumulative incidence of Covid-19 cases between the placebo group and the vaccine group. It clearly shows that the cases of Covid-19 increase regularly in the placebo group, unlike the vaccine group. It takes about ten days for the vaccine to effectively prevent the onset of the disease and the second dose is also essential. Regarding the prevention of severe forms, there were too few in the vaccine group to conclude on the benefits of the vaccine on this specific point. It is also not known whether people vaccinated are still contagious or not, this has not been tested during these clinical studies.
Despite the record time with which it was developed, Pfizer-BioNTech's Covid-19 vaccine is effective and well tolerated. Even if some questions remain, the risk-benefit balance seems to be in favor of profits. The pharmaceutical company filed an emergency use request on November 20 with the FDA, which has yet to render its verdict. For Europe, the European Medicines Agency started the evaluation of Pfizer's vaccine on December 1st.
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I need this very much because I have no experience on it. So I want to publish an article by using this kind of web based tools.
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Use the above link, it is perfect to build a transcriptome profile either by commands for a certain cell line (with real RNA-seq data), or just using the user friendly website. It is also possible to address comparisons between cell lines by applying the proper ratios, like GeneA/GAPDH in cell line X vs GeneA/GAPDH in cell line Y, using FPKM or TMP values.
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I am working with frozen single cell solutions of human glioblastoma samples. However, upon thawing I see 90% debris and dead cells and only a tiny fractions of live cells.
I have tried Miltenyi microbeads, but they are super oversaturated with all the "dirt" and I see almost no improvements.
Now I do flow cytometry based sorts and have SYTOX Red in there for viability.
However, the cell fraction is not pure yet.
Do you think adding Calcein AM would improve? Or do you have a different suggestion?
I am looking at single cell mRNA downstream as a readout and need the cells pure and vital.
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I agree with the Malcolm Nobre's reply. You can give a short centrifuge in order to pellet down the live cells and remove the sup. (containing dead cells and debris). Good luck