Science topic
Transcription - Science topic
Explore the latest questions and answers in Transcription, and find Transcription experts.
Questions related to Transcription
Hi everyone, I am working on in-vitro transcription to generate mRNA for LNP fabrication. The kit I am using is the HiScribe® T7 mRNA Kit with CleanCap® Reagent AG from NEB. The first time I was doing it in October, the yield was very high. But the yield has been getting lower and lower since the second time.
Even with the control template the kit provided, the yield is always lower than 7 µg/20µL reaction.
Then, the technical support sent me a new kit, but the control template generated an even lower yield.
I've been talking to the technical support for a whole month. I am sure that I always put the T7 polymerase mix on ice, add reagents in the same order as the protocol, and set the reaction in room temperature. I am always aware of the RNAse. There was no RNA degeneration diffuse under my RNA band. The electrophoresis always shows very fainted bands even from the aliquot of unpurified 20µL IVT reaction.
Now, I suspect that the enzyme just started degenerating since I opened the T7 polymerase tube. Does anyone encountered this situation before?
I have a list of genes and would like to find transcription factors that regulate them based on experimental evidence. I have used some online tools, but they provide different results. Can anyone recommend a reliable tool or database for this purpose?
Thank you!
I want to find out different isoforms of a gene. I know microarray or RNA seq can be used. But I need clarification or any other methods that can be used.
I found the phosphorylation level of ERK1/2 in my cell sample increased when I treated the cell with a medicine, but the mRNA level of my target protein decreased obviously. Is that normal? Coz when ERK is phosphorylated, it usually bind to the transcriptional factor and promote transcription, right?
I created a stem cell cell line with BCR-ABL1 fusion transcript. The Sanger sequencing at DNA level confirms fusion but could not be detected by RNA seq. The fusion in DNA level is located at the intronic region for both BCR and ABL1. Cell growth is different (higher) in the mutated cell ine with BCR-ABL1 fusion compared to The wild type counterpart. This is very likely because of the presence of such a cancer fusion gene. Is it possible that the fusion transcript is not possible to be detected in this case?
Generally, CRISPR-Cas9 editing leads to the formation of indels at the target site in the coding sequence of a gene. This often results in an altered reading frame (frameshift mutation) which will then lead to the formation of truncated/ non-functional protein. So, in this scenario, transcription should continue as usual and we should see a similar gene expression level as WT in the qRT-PCR. There is no doubt that transcripts will produce the truncated/ non-functional protein. Am I correct?
Since i already have factors that im studying. and im using Quanti quali as my approach. Can i precode factors in Atlas.ti, upload transcripts and look for supporting quotations to the results i obtain in quanti?
Just sharing my experience of doing thematic analysis for my in-depth interviews conducted:
When conducting in-depth interviews for a qualitative study on school stakeholders, i tried to compose the responses in spoken language -verbatims and then to transcripts. Well ,with a little of my experience of qualitative research, can suggest a few approaches handle large datasets in qualitative studies, please note that it requires the following for effective management:
- Guys methodically classifying unprocessed data (e.g., coding transcripts in qualitative data analysis programs such as Atlas.ti or NVivo)- I have personally used Atlas ti and personally loved it. (becos of the web version)
- later considered dividing data into digestible categories using early coding frameworks.
The next challenging phase needs more focus- please note how it was handled..... The categories were iteratively refined to prevent redundancy and data saturation.
Dear all,
I am trying to label TPM3.1 in live cells. I was wondering if there is a Sir-Dye specific to this ? if not, is there an existing plasmid designed for IVT (mRNA) of TPM3.1-GFP or RFP or else tagged which I could inject to my cell?
Thank you in advance.
When I used 95bp PCR product (full-length tRNA with promoter), the T7 polymerase RNA synthesis kit (NEB) gave a lot of large bands (more than 200bp). Can you explain the reason and the way to remove all the bigger RNA bands.
Hello, according to published papers: https://www.nature.com/articles/nprot.2013.143/figures/4
It is recommended to have an extra guanine before the 20 bp gRNA sequence, for my efficient transcription from the U6 promoter. If my gRNA already starts with a 'G', do I still need to add an additional guanine?
I was just wondering as we can (quite easily?) isolate both RNA and proteins from the same sample, why can't I find much info about sequencing transcriptome and proteome "at the same time"?
It seems that single-cell multiomics is in trend now, but looking at transcripts and proteins from the same samples looks like a simplified multi-omics from my perspective. What are the limits to that? Even companies don't seem to provide such services. Why is that?
Hi everyone,
For a qualitative research study, we are scoping for potential transcription software to transcribe our interviews. We have the following criteria in mind:
- We are looking for affordable software (maximum cost of $200 for 16 hours/960 minutes of transcripts).
- Software should be good at transcribing East-African accents in English (specifically, Ugandan accents).
- Software should have high data protection mechanisms in place. At minimum, it should be compliant with GDPR legislation.
I already came across Otter.ai, Trint, Sonix.ai, and Rev.com. I am wondering if you have used any of this software before and can provide feedback? Other suggestions for software that meets the aforementioned criteria are also welcome.
Thank you in advance for your responses!
What instruments might measure empathy of adult students in asynchronous online learning discussions? We would have access to the transcripted text of academic conversations between peers without having access to the students who engaged in those discussions. Thanks for your ideas!
I would like to know if T7 in vitro transcription can be effectively terminated by pol II terminators (specifically bGH poly (A) signal)? And in reverse can a pol II promoter (eg CMV promoter) induced in vivo transcription be effectively terminated within eukaryotic cells by a class I terminator specicially T7 terminator (TΦ large terminator)?
The target transcript was clearly detected in my Northern blot results. Howerer, two " white" bands which are the same band sizes and locations as 18s and 28S rRNA were shown, what's the reason for this? thanks in advance.
It is said that the transcription is started by using a start codon.. my question is that why all those are necessary?
I was wondering if it is possible to form a permanent open "ssDNA bubble" similar to a transcription bubble (>13 nucleotides) within E. coli. These criteria are important:
1. Open ssDNA bubble within replicable (in E. coli) genetic element. So no C-Traps under force.
2. No proteins, nucleic acids, or other toxic chemicals supporting the bubble. Can help during nucleation, but bubble has to be accessible for protein interaction.
3. Stable in bioorthogonal conditions. Physiological pH, salt, 37 °C, etc.
What recording and transcription apps/tools are best for historical research? I will be conducting interviews of about 60 - 90 minutes.
TIA
Hi everyone,
I have DNA templates for T7 IVT mRNA transcription that are between 250 and 1050 bp long. I want to run T7 IVT mRNA synthesis using NEB2080 kit as recommended, but since tempates are so small, would you recommend to run IVT incubation overnight? For how many hours?
Any other recommendations would be greatly appreciated.
Thanks!
Kind regards,
Maria
As the title described, it is common that a target gene sequence contains motif that acts as the binding site of transcription factor rather then the upstream regulatory region. Does this affect gene expression? or transcription factor just release followed by subsequent upstream transcription? please share your thoughts.
Hi Everyone, I'm conducting research on culture shock using a mixed method design (Questionnaire + Interview). I have 60 Participants in Questionnaire and 10 participants (out of 60) in the interview. I have conducted my research regarding different factors causes culture shock (total 7 factors ). My question is that when writing the analysis section of my PhD Dissertation, how many interview participants should be quoted/mentioned in each factor analysis ? Is it appropriate to mention/quote the interview transcript of all 10 participants in each section or should I just mention/quote 3-5 participants? In most PhD Thesis that I have seen, the author mentioned/quoted a maximum of half of the participants.
I was reading the T7 RNA polymerase protocol and noticed the following sentence regarding incubation time: "Incubate at 37°C for 1 hour. For shorter (< 300 nt) transcripts incubate at 37°C for 2–16 hours."
Can't think of a reason why short transcripts would take longer than long ones. Any ideas?
I will perform a apoptosis evaluation in cell lines using flow cytometry after siRNA transfection, and I want to evaluate the levels of BAX and BCL-2 to complement the experiment. However, I can only do a qPCR of both transcript, but not a western blotting. Can qPCR be used in this case, or it would be only informative if I measure the protein levels?
The desired gene region was amplified by PCR and the resulting PCR products were cleaned. Bands were observed in an agarose gel and measured in a spectrophotometer. Then, transcription was performed with the ABm Onescribe T7 transcription kit (E081). Of the PCR products, 196 ng for gene 1 and 133 ng for gene 2 were included in the transcription reaction. Despite the assurance that the kit was functioning correctly, the transcription products did not yield bands in agarose gel electrophoresis. No bands were observed with or without the optional DNase treatment following transcription in the protocol.
1. What could be the cause of the transcription problem?
2. What are the alternative methods to prevent DNA loss in the clearance of PCR products despite increasing the initial concentration?
3: How can the formation of dsRNA after transcription be determined? Can it be visualised with agarose gel electrophoresis? does the PCR product and the transcription product give the same gel image? Can it be measured in a spectrophotometer and what is used as a blank?
I am working on isolating Naive CD4+ T cells from human PBMCs using the Miltenyi AutoMacs. However, I am obtaining a higher percentage of Naive CD4+ T cells from the PBMCs than expected. I would like to confirm the identity of these isolated cells using transcriptional primers for mRNA gene expression analysis. Are there any specific primers that I can use for this confirmation?
Thanks!
I am planning to do invitro transcription and capping for an mRNA vaccine generation. Can anyone suggest whether it should be done on a bench top or inside a laminar hood?
I am performing PCR as a QC test to look for a transcription gene that should be negative after a CAR T therapy process. As we are comparing against a CAR transduced patient's cell, we require a used transduced ATCC cells. However, the ATCC cells have a low transfection titer, which makes the PCR band faint and when kept for long, it becomes fainter and fainter.
I was thinking of using another different grade of cells such as transduced research grade cells as it was observed that the bands tend to be much brighter than the ATCC grade cells.
Is it possible to use transduced research grade cells instead of ATCC grade?
Hi Guys
any Repository of Qualitative interview transcription??
Hello everyone,
I am working on the production of IVT mRNA. After transcription, the remaining DNA templates are digested by DNase I. I have tested transcription with uncut DNA (mostly supercoiled) and with linearised DNA as it is poorly described in literature, how the efficiency of transcription is reduced by using supercoiled DNA. What I ended up finding upon viewing my transcription products on agarose gels, was incomplete digestion of the DNA when it was in its supercoiled form. So I am now taking a closer look at that. Can anyone advise how the form of DNA impacts the efficiency of DNase I digestions?
Greetings, dear colleagues!
Our team conducts research on newly discovered SIRC elements in plant genomes ( , which are thought to be MITE transposons losing inverted repeats products, which could influence genome regulation) using bioinformatics, and we plan to conduct experimental molecular biology studies to elucidate the functions of SIRC. The problem is - our team is specialized in molecular bology experiments aiming to reveal the functions of genes, not non-coding DNA elements. That's why I want to ask your expert opinion - what experimental techniques would help to reveal the functions of abundant DNA elements of repetitive nature?
What comes to mind is the creation of mutant lines without several of these elements, but such experiments are too large-scale and can last for years, which is too complicated at the moment.
Another technique that comes to mind is the amplification of certain sequences and examination using circular dichroism spectroscopy to reveal whether given elements have unusual secondary structure like G-quadruplex of triplex DNA etc that could influence processes of genome transcription or replication.
And one more - we thought it could be possible to capture and identify plant proteins that specifically recognize SIRC via some modification of EMSA (electroforetic mobility shift assay) method. Unfortunatelly, up to date we didn't find any mentions of EMSA variant that uses not single purified protein, but whole DNA-free nuclear lysate, with subsequent identification of binding proteins via MALDI-TOF.
What other in vitro experiments could be useful?
What are the key differences and benefits over using CRISPR dCas9 system /CRISPR interference (CRISPRi) for transcriptional regulation over more traditional methods such as Structure-based combinatorial protein engineering (SCOPE) or others?
During my analysis, I observed that genomic position 77 is annotated with gene symbols F, M, NP, and P across various transcripts. Please find the example below.
I used the gff3 file from NCBI https://www.ncbi.nlm.nih.gov/nuccore/AF077761 for variant annotation that includes details about gene symbols and transcript types. But I'm not sure what it means biologically to have different gene types at the same position. Does anyone have an idea on this? Please shower some ideas.
For example
NODE_30_length_153_cov_24.473684_77_-/C NODE_30_length_153_cov_24.473684:76-77 C gene-F rna-F Transcript upstream_gene_variant - NODE_30_length_153_cov_24.473684_77_-/C NODE_30_length_153_cov_24.473684:76-77 C gene-M rna-M Transcript upstream_gene_variant - NODE_30_length_153_cov_24.473684_77_-/C NODE_30_length_153_cov_24.473684:76-77 C gene-NP rna-NP Transcript 5_prime_UTR_variant 21-22 NODE_30_length_153_cov_24.473684_77_-/C NODE_30_length_153_cov_24.473684:76-77 C gene-P rna-P Transcript upstream_gene_variant -
I have gel images of the plasmid that has been digested with various restriction enzymes and an image of the transcription of digested plasmid and a plasmid map with the cut sites located. I understand that the smallest transcription product on the gel is closest to the promoter. I get a band of transcribed RNA approx 100bp with EcoR1 so I know that on the plasmid map the promoter is either 100bp upstream or downstream of the EcoR1 cut site but how do I then know the direction of transcription.
I recently encountered an intriguing situation while examining a plasmid constructed by someone else for a eukaryotic expression system. This plasmid contains a unique arrangement of open reading frames (ORFs) that has sparked several questions regarding the potential outcomes of their translation.
In this plasmid, there is an ORF near the 5' end, where the translation initiation site is quickly followed by a stop codon, potentially resulting in a very short peptide. More interestingly, nested within this first ORF is a second ORF that begins inside the first ORF and could potentially translate into a much longer protein, consisting of 500 amino acids.
Given the common understanding that eukaryotic transcripts typically feature a single ORF, the discovery of this arrangement has led me to ponder the following questions about the translational dynamics in this specific scenario:
- In the context of this plasmid, will the translation machinery be capable of bypassing the short ORF to translate the longer protein, or will it prioritize the translation of the short peptide due to its proximity to the 5' end?
- If both peptides are indeed translated, what might be the expected ratio between the production of the long and short peptides?
- Is there a possibility that only the short peptide will be translated, effectively ignoring the translation potential of the longer, nested ORF?
Furthermore, I'm curious about how this scenario might differ if the plasmid were used in a prokaryotic system, which is known for its ability to translate multiple ORFs within a single transcript.
I'm seeking insights, experiences, or any relevant literature that could help shed light on the translational strategies employed by cells when faced with plasmids containing nested ORFs, especially in the context of eukaryotic expression systems.
Thank you in advance for sharing your knowledge and experiences.
Hi ALL,
I am using a pair of primers to amplify a region in my gene of interest from cDNA samples. The cDNA samples are extracted from tissues of mouse of different ages. The gene is known to have decerased expression level when mouse ages. However, I did not see any change of the RT-PCR amplicon band intensities on agarose gel, indicating no change for the transcript level. I did not saturate the PCR products as I tried different cycle numbers (from 23 to 30 cycles). What could be the possible reasons? Should I design new primers targeting a different regions in my gene? Thank you for the help!
By using CRISPR-Cas9, we can insert any sequence into any locus we want, right?
Base on that,
What I'm actually curious about is that,
Is it possible to regulate the transcription of endogenous gene by inserting short sequence ( for example, binding site of specific TF ) at TSS-proximal region?
I'd be appreciated to hear examples of any studies that have a similar concept to "put a short sequence upstream of a gene".
Thank you for your interest in my question :)
What tools or methodologies are available to comprehensively analyze RNA expression profiles across various human tissues, specifically to discern and compare transcript variants of a gene
We are looking to confirm transcript expression in cells that are positive for a reporter gene (tdTomato) in mice. We are able to locate the transcript in question via RNAscope. However, following the RNAscope protocol, we are unable to locate *ANY* tdTom neurons.
We know that tdTom is expressed in these mice, as we are able to visualize tdTom neurons in alternate series that were not exposed to the RNAscope protocol. Thankfully, the observed tdTom neurons overlap with transcript expression in alternate series. however, we need to be able to colocalize these signals with cellular resolution.
Any experience, suggestions, and/or references would be greatly appreciated.
C
I have cloned my genes in a CMV vector and transfected them in HEK293 cells. After harvesting the cells, the expression level at the transcriptional level is increased compared to the control well. However, the peptide sequence is not seen to be expressed as analyzed by mass spectrometry.
Can you please give an insight into the possible reason for the same?
Thank you.
Hello!
This is my first time analysing qualitative data and so I have chosen to use QDA miner. I have six transcripts to analyse.
I have watched multiple tutorials on YouTube on setting up a project and followed them exactly. However, when I select the files first of all I noticed I am unable to click the remove text formatting option. The box is there but I cannot select it. Then when I import the documents they all have no text in them. The file names have imported but they are blank
I have tried converting my files into pdf and rtf versions in case this helped.
Any advice welcome!
I use the MEGAscript T7 Transcription Kit (Cat. no.: AMB13345, ThermoFisher) for in vitro transcription of linearized plasmids. However, I have measured a high concentration of DNA in my mRNA after in vitro transcription. I use 15-20 ng of plasmid for the reaction, and I have already incorporated a second DNase treatment step in my protocol, but it doesn't seem to remove the DNA. I use DeNovix for measurement of concentrations. Anyone who has experience with this? If yes, is your mRNA still functional even though DNA is present?
I would like to design a few couples of primers to amplify several define transcript region of IgG gene. With forward primers in the V or J region and reverse Primers in the constant region. However, I can not check the primer specificity using NCBI primer blast. Do you know which platform or sofware that I can do primer blast for IgG transcript? Thanks and regards.
- Observational Analysis: Researchers observe and record conversations to study how individuals from diverse linguistic backgrounds interact.
- Transcription: Spoken language is transcribed into written form for detailed analysis, including pronunciation, intonation, and pauses.
- Coding and Categorization: Linguistic patterns and sociolinguistic variables are identified in transcripts, such as code-switching and language choices.
- Quantitative Analysis: Statistical techniques may quantify sociolinguistic phenomena like code-switching frequency or linguistic feature distribution.
- Qualitative Analysis: Researchers explore the meaning and context behind linguistic behaviors and language choices.
- Questionnaires and Surveys: Self-reported data from participants, including language preferences and attitudes toward languages, can be collected.
- Corpus Linguistics: Large collections of texts or spoken data are analyzed to uncover linguistic patterns.
- Experimental Studies: Researchers design experiments to manipulate variables related to peer interactions and sociolinguistic competence.
- Interviews: Semi-structured interviews provide insights into participants' experiences and perceptions.
- Audio and Video Recordings: Recordings capture spoken and nonverbal aspects of communication, such as gestures and facial expressions.
Hi everyone,
I got an exon level counts matrix (obtained via the Bioconductor recount3 package) and I would like to transform the exon counts to an estimation of transcript abundance.
Does anyone knows a way of doing this?
Hello,
We have a gene that is modulated by the binding some transcription facors (TFs). We recently found that one of these TFs is mutated and we want to know if this TF can still bind to that gene. Is there any software or tool to figure this out?
Thank you in advance.
from where the transcription of the gene in the vector starts , is it always from 5 to 3 or it depends on the direction of the promoter .
I did a knockdown, after checking the level of protein expression with Western blot, I got some percentage let's say like 60 to 70 % of knockdown, but when i tried checking with qPCR to check the transcription level it was as if there was a complete knockout, what could have been the reasons, I am confused can some help me with an explanation? You help will be highly appreciated.
Analysis of qualitative data requires intensive reading of the transcripts, field reports, diaries, journals, and other documents. It is a continuous and to-and-fro process. What changes do you, as a qualitative researcher, face during data analysis and how do you overcome those challenges?
Dear colleagues,
I'm currently running some in-vitro transcriptions with the HiScribe T7 RNA Synthesis Kit by NEB. As Templates I'm using a purified PCR product as a run-off template. I properly thought about my Promoter sequence, bearing all the necessary bases not only for the binding but also for the initiation site. I'm expecting a ssRNA of roughly ~750-780 nt for my transcripts. However, my Urea-PAGE looks like displayed here. I can't really explain all the additional bands...
After the in-vitro transcription I'm using a RNA clean up kit and purify my transcript prior loading on the gel. I don't think it's unspecific degradation, the band look to sharp for that.
I'm looking forward to your suggestions and expertis

I know many websites have simple tools like transcription and translation available, but are there any analysis tools that researchers need that either do not exist or are not publicly available? It could be anything from algorithms to visuals. Thanks!
can a DMRT( Duncan's Multiple Range Test) result in 11 columns for which i get compact lettering till 'k' (starting from 'a')?
Software used : SPSS v25.0
After i did the post-Hoc with Duncan, i got an output for a specific dependent factor with 18 treatments (3 transcripts of each) as 11 columns.
To put this DMRT result as compact letter display in my table, i'm getting letters 'a' to 'k' for the respective columns.
Is this possible/correct?
significance values for few columns exceeds 0.05. and few are below 0.05.
I'm working on a qualitative study that will include 30 interviews conducted online in Arabic. Can you please recommend reliable transcription software that supports Arabic? It doesn't have to be free
Get more than 80% - 90% of transcription efficiency
Transana is a software for qualitative research that enables researchers to work on authentic data that is recorded or videotaped. If anyone has employed this tool, I would be interested in learning more about their experience with the tool and its effectiveness in managing and analyzing complex qualitative data in their studies. Furthermore, the software is with different versions, so I am confused about which one to use.
I want to produce 29kb mRNA using the in-vitro transcription method. It will be used for protein production in mammalian systems. I found RiboMAX large kit that can make a 27kb transcript. However, I did get any information about its application on protein production in the mammalian system. Could anyone suggest the best method or kit to produce this long-size mRNA transcript?
Hi all, I am trying to extract RNA from 12 well plate and so far the yield obtained from trizol/chloroform protocol is OK.
After that I tried to do DNAase step (using NEB DNAse I) but I found that it does not allow cDNA transcription.
I don't know if it was the inhibition step 70°C x 10min of DNAase activity or smth else.
Do you have any similar experience? if yes, any trick to share with me that can help to do the DNAase step without inhibiting the reaction downstream? Thanks a lot
Dear all,
Thanking you in advance for your attention, I would like to ask you two questions regarding an electrophoretic run on 2% agarose gel:
1) I did an electrophoretic run of the Real-time PCR products (with Sybr green). Although the band of my target product is evident, I notice some very slight sub-bands (indicated by the arrows) which, according to the molecular scale, could have a size of 500 bp. I designed the primers by myself, and making a blast they are specific for my gene of interest . What do those sub-bands represent? Can they affect the analysis of fluorescence?
2)The same primers gave me products of a different size (in the photo, the circled bands). The primer targets a gene that has 3 transcript variants. Each well has a different individual (animal). Could those two circled products be a transcript variant?
Thanks a lot for you precious help,
Best Regards
Matteo

In allotetraploid B.napus plant transcriptome, Based on pacBio 3rd generation sequencing, one gene has evidence of Alternative splicing (AS) in it. This gene has four exons and produces 2 distinct transcripts. The structural analysis of two transcripts shows that transcript 1 is produced by using only exon 1 and 2 of gene, whereas, the transcript 2 is produced from exon 3 and 4. Therefore, these two transcripts have no overlapping sequences between them. I have to verify the presence of these two transcripts. Is there any other other strategy to amplify the these two transcripts at once and show the evidence that these two transcripts are definitely transcribed from the specific gene, except using the transcript specific primer for each transcript (using transcript specific primer will just help me detect that transcript but will not provide enough evidence that it is originating from this specific gene.)?
"during both the sample preparation and computational analysis phases, at which imperfections and biases may be introduced. These limitations may affect the ability of the experiment to address specific biological questions, such as correctly identifying and quantifying which of multiple isoforms are expressed from a gene8 . This example is particularly relevant to very long, or highly variable, transcript isoforms such as those found in the human transcriptome; 50% of transcripts are >2,500 bp long in humans26, with a range from 186 bp to 109 kb" the Author of the Artical
RNA sequencing: the teenage years - PubMed (nih.gov)
31341269
transcription and translation of eukaryotic and prokaryotic cells
Hello,
I am designing a plasmid with an SV40 promoter-driven antibiotic resistance. Does expression from an SV40 promoter require a TATA box upstream of the transcription start site? The original vector had a TATA box at -30, however this is lost in my cloning strategy. With my current plan, the transcription start site is just 8bp from the end of the SV40 promoter. Will this allow for expression, or is a TATA box needed?
Thanks!
I am working on the transcription of an RNA strand. More than knowing all the characteristics of my RNA, I would like to know if the transcription was successful or not. What methods can be used for this? My RNA strand should be 21nt long. Is it possible to use gel electrophoresis or spectroscopy?
Hi! I'm planning on using methyl-3-nitro-1-nitrosoguanidine (MMNG), for inducing transcriptional mutagenesis. From my understanding MNNG causes the formation of O6‐methyl guanine (O6MeG) inducing thymine mispairing during DNA replication. However, is it likely to for MMNG to to lead to other mispairing that may occur in low abundance? Or are there any disadvantages to be mindful of? I want to ensure that I can accurately quantify mutation patterns.
Thank you for your insight :)
Can anyone recommend a software that could be used to help in Arabic interviews transcription/ translation? I am currently using Trint, but unfortunately it is not accurate.
I'd like to know that what are the different ways to know/identify whether a particular Gene is expressed or not ?
Few points from my side are :
1) identifying it's corresponding m-RNA transcripts level.
2) identifying the protein that was produced by the expression of that particular Gene.
Any other points ?
I was reading a research article where I found this term but unable to understand. AK5 gene in 2009 was reported to have two transcript variants i.e. AK5p1 and AK5p2.
in transcription process why doesnt need primer for bulid mRNA
Hello everyone!
I have interesting question asked by my professor and I could not find relevant answer anywhere.
Why are we seeing up and down pattern on transcript abundance? Example RNA seq data for a gene from a rice transcriptome data base is attached. LOCUS ID is highlighted in yellow and transcript abundance is in below three samples after drought treatment.
The question is ,why the signal level is not uniform on Exons? is it low signal reads? Why there are gaps or sudden fall in signals? ( which are Marked in Red arrows) How to read and understand this? and I know this is the common pattern in RNA-seq data, but I don’t know why?
It’s an interesting question asked by my professor! can any bioinformatician help me understand this? Thanks in advance.

Hello scientists, I was hesitant to ask such a question because it is somewhat simple, but in the end there is no shame in the learning process, so, What consumes more energy, the polymerization of coding region or its translation?
and how to calculate the energy required for transcription?
Thanks in advance.
I performed an RT-PCR on my gene of interest hoping to see which isoforms are present on the mRNA level. Literature suggested there should be 4. To my surprise, i see lots (too many to count) of transcripts of various sizes. I isolated about 50, sequenced them, and aligned them to the original gene cDNA. Majority of the transcripts I isolated have chunks of sequence missing at seemingly random places, with random chunks of exons would be missing here and there. some has majority of exons missing.
I am wondering if transcription often produce these aberrant transcripts or is something unique going on here?
I will be interviewing clergy members.
Why are mouse chromosome Y transcripts (avg) significantly shorter than its other chromosomes' transcripts? The calculation & comparison of the average lengths were done with t test according to the entire UCSC mouse genome. Any ideas?
Hi to all.
My question is how can I optimize my RTqPCR if the cDNA dilutions ended up in similar Cq?
I synthesized my cDNA from 350 ng total RNA, assuming 1:1 production I should have 350 ng cDNA in 20 ul right? Then I did a dilution of 1/2, 1/5, 1/10 and 1/20 (I know the first three are consider quite a lot to be used in the run) and used them in a 20 ul run. The gene is a ref. gene: GAPDH. Interestingly the Cq values aren't that different between the dilutions (~29, ~30, ~31 and ~30). Obviously these aren't good values but I don't know what can I do to optimize the run.
I’m going around in circles trying to find anything on APA (7th) recommendations for formatting supplementary materials such as an interview transcript. I am a student so one of the general recommendations for a student paper is double spacing for instance, but in an interview transcript, which is over 10 pages long with single spacing, I’m afraid the document will be unnecessarily long and hard to read. Are there specific rules regarding formatting an interview transcript?
While trimming the adaptor and low quality RNA-Seq illumina paired end reads in Trimmomatic, I have got more Forward only survive of about 40 to 50%. This study is for estimate the transcript abundance (DEG) at various condition. How is the possibility to continue further...
1. USE singleton reads (R1-For only)
or
2. Only use both paired (survive) high quality reads (50% of the reads)
Any suggestion, Thanks in Advance
by, Ellango R.
I am working with a non-model organism and we generated a complete genome that is annotated. How can you determine the transcriptions start sites in this genome? Is there any bioinformatic way to do it (software)? or Do I need to do an experiment to identify these sites in the genome? I know that CAGE seq and CHIP-seq are good techniques to do that. But I am not sure if there is a computational tool to identify these sites.
Hi there,
I'm trying to design primers to detect CRISPR-mediated KO using qPCR (as described in this paper: ).
I designed a few pairs of primers and neither of them show any signs of amplification.
As an example, this is a pair targeting the ITGA2 gene (exon 2):
Primer_FWD ("watching"): ATTGTTGTTTGGCCTACAATGTTG (target +/ 53026780)
Primer_REV: CTGCATAGCCAAACTGTTCACT (target -/ 53026816)
I used the following transcript for the design:
Imported from genome: GRCh38 (hg38, Homo sapiens)
Gene: ITGA2 (ENSG00000164171)
Location: chr5 52989326-53094779
Transcript: ITGA2-001 (ENST00000296585, CCDS3957)
This specific pair is a little low on GC%, but others have GC% within the recommended 40-60% range. However, neither primers seem to be working.
Apart from GC%, these primers comply with the recommendations for PCR primer design published on various websites. Yet, something isn't right - on qPCR amplification curves are dead flat after 40 cycles (I've tried a few template concentrations and annealing temperatures with no success).
Would anyone have some suggestions on what I do wrong? And what can I try?
Thank you in advance.
What are the best assistive software that you're using to facilitate the transcription of qualitative data? I use soundscriber (a free tool). I am looking for better options.
Thanks.
I conducted data collection (20 interviews in Sepedi Language) as part of my Ph.D. studies. All interviews were transcribed then translated to English.
So i would like to know that out of 20 transcripts how many should i back-translated to Sepedi to ensure/check accuracy?
Any literature recommendations will be appreciated
Can someone recommend a plasmid that carries two transcriptional terminators ? We'd like to insert two adjacent terminators into a construct we have made to prevent any transcriptional readthrough. We are currently using one lambda oop terminator, but we'd like to improve upon this, if possible.
Unfortunately, we are unable to synthesize tandem terminators by gblock due to their structural complexity (IDT won't make them) and we have had problems amplifying terminators by PCR likely for the same reasons.
Our ideal strategy would be to cut an clone a DNA fragment bearing two tandem terminators. Any recommendations?
Hi all,
I'm currently in the process of working on DIG-labeled mRNA probe transcription. I've done this successfully plenty of times, but the process never fails to create new ways to stump me, and I'm having trouble solving this one. I ligated my fragment into a PGEM T Easy vector and digested with SpeI for T7 and SphI for Sp6. I checked to make sure neither recognition sequence is in my fragment. I sequenced my plasmid after miniprep, and the sequences were perfect. The digest looked fine (photo attached) with the SpeI and SphI fragments both at the same, correct size. Only weird part is that the uncut plasmid ran slower that the cut plasmids, which has never happened. Issue persisted with replication. But that's aside the point. My PI told me to transcribe anyway since the cut plasmids were all the correct size.
I first transcribed last week, and the T7 looked perfect. Sp6 did not. I know that RNA can take multiple conformations, but I've never seen the bands look like this when that happens. Our T7 polymerase is pretty new, but Sp6 is a bit older, so my PI had me order a new tube. I tried again yesterday, re-transcribing both T7 and Sp6. T7 still looked perfect, but Sp6 did the same weird thing, just more intensely. It's hard to see on the gel because the second Sp6 transcription is so bright, but the bands are the same sizes for the Sp6 probe on both runs.
If it's a case of multiple conformations of RNA, I don't really understand why only Sp6 would be displaying it in both rounds of transcription. I have a feeling that my PI will suspect issues with the restriction enzyme and have me order more, but I want to explore any other avenues.
Any thoughts about what's going on here will be greatly appreciated.


We have a gene with two isoforms, one with a longer 5' UTR and one with a shorter 5' UTR. It's been demonstrated that which isoform is predominant changes over development.
We want to study how the UTR might influence the translational efficiency of the transcripts. In the past, we tried to create a luciferase fused construct that had our UTR, our gene, and luciferase. However, the results were somewhat messy and hard to interpret and we questioned whether the luciferase construct itself was affecting translation.
Does anyone have any experience with alternative methods of studying 5' UTR's effect on translation, or have better experience with luciferase constructs?
I recently did a transcriptome assembly using Trinity and I got one Fasta file. I want to do further analyses on unigenes only. My question is how do I identify the unigenes from the transcripts and have a Fasta file of unigenes only?
I have been trying to get RNA via in-vitro transcription of a G-rich DNA sequence after PCR amplification since 5-6 months. But i fail to observe any RNA bands under UV light after running the in-vitro transcription product in Urea PAGE. Sometimes I observe a faint or a smeared band after EtBr staining. Can I get any suggestions about the same?
Dear community,
does anybody have experience with cloning two genes in opposing reading direction so that the genes theoretically could share just one bGH polyA signal.
I think the inverted polyA signal for one of them should not make a problem, since the polyA has no "direction". I am just concerned that the polymerases might sterically "crash" when both genes are transcribed at the same time but wouldn't that also terminate the transcription? :D
prom->tDTomato->bGHpoly(A)<-PuroR<-prom
Best!
I am not able to visualize anything on agarose gel except primers and gene ladder.
Hi everyone,
Please correct me if the information mentioned here is incorrect. In the animal mitochondrial genome, there are no introns coding sequences.
I was wondering whether the primers designed for a mitochondrial gene (DNA sequence) work for the cDNA for its mitochondrial transcript?
Looking forward to a discussion!
Thanks in advance.
I have a big problem in my transcription. After taking my cDNA and PCR of my gene of interest, I did purification and miniprep. My results shown everything are great (nanodrop and agarose gel). But I can't have my RNA. In my agarose gel, I see a trail.
Why is there a bright smear below my RNA band from in vitro transcription?
I have only observed it for my longer transcript.
I want to publish the (qualitative) data that is associated with my research paper and have explored journals like Data in Brief and Scientific Data published by Elsevier and Springer respectively but they could not consider the data (descriptor) manuscript I submitted to them due to technical and situational reasons. Other journals I have seen are not suitable as many of them either consider other types of data or are based subjects different from mine. The data based on social research and consists of the interview transcript on pandemic policing and public (compliant) behaviour.
Are there any bioinformatics tools or software available that allow verifying if the processed transcript acts as a lncRNA?
It is about Molecular Biology. Please answer up. Thanks
The primers designed should be allele specific so as to amplify either my WT or Mutant transcript and not both.
I've been trying to do invitro transcription of 70 bp long DNA oligo using T7 RNA polymerase to figure out formation of G-quadruplex structure. The following components are added to the master mix for 20uL reaction volume:
1. 200nM ds DNA (prepared in 50mM tris and 10mM MgCl2),
2. Transcription buffer {trisCl (pH 8), 2mM spermidine, 50mM KCl, 10mM MgCl2, 10mM DTT} followed by
3. 40% PEG 200 and at last 2mM NTP .
I have successfully carried out transcription using this method earlier but now gradually the transcription seems to be having an issue.
The problem is when NTP is added to mixture having peg 200 , it immediately forms white precipitate. I tried repeating this in absence of peg and seen that there is no ppt formation. Not able to understand why is it happening. Is there any report of interaction of peg 200 with NTP?
After EDTA addition, the precipitate disappears. To check if DNA is precipitating due to Mg2+ chelation, I have also made the reaction mixture in absence of DNA , still I can see precipitate formation.
Can someone please help.
Note: I have assembled the reaction in room temperature.
Thanks in advance.
Hello,
We are currently evaluating software for the analysis of transcribed video recordings of dialogues. Coding units are episodes, and we want to quantify codings as duration of time. In previous studies, we successfully used Transana for similar analysis.
We are now evaluating to use MAXQDA instead, which does not assist reports of time duration of codings on transcripts, but only number of characters. For the export of duration data, coding has to be on the video, which does not meet standards for linguistic discourse analysis.
Does anybody know studies and/or have experiences with number of characters instead of duration as value for the quantification of transcript-based coding of dialogues?
Thank you for sharing your knowledge and experience!
Kind regards, Annelies Kreis
Im using mMESSAGE mMACHINE T7 ULTRA Transcription Kit to transcribe my DNA. It produced 30 ng/ul which is 750 ng although the kit promises to yield atleast 15-20 ug of RNA. Even the control DNA provided in the kit produced 6.2 ug RNA. I followed the protocol as it was instructed. Kindly share tips to increase the yield upto 10 ug atleast. TIA!
I am trying to express a short RNA sequences in the nucleus in Drosophila. Are there any UAS-based expression vectors in flies that do not have PAS in the 3'? Or on the other hand, is it possible to just express a transcription termination sequence at the 3' of my transcript so that my transcript terminates before reaching the PAS? I don't want to have to mess with the vector backbone itself (removing PAS...etc.). I am quite new to molecular work regarding vectors, so if I am missing something important, under a misunderstanding of sorts, or if you have good suggestions, please let me know. Thank you!