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16

It has been well established that mRNA abundance serves as a poor proxy for protein abundance in most cases. This paper on yeast and this paper on cancer both establish this, although using older techniques (SAGE and microarrays, respectively), while this more recent review discusses the topic in light of more recent technologies (e.g. RNA-seq). Perhaps the ...


12

I don't have any literature to back this up but I doubt that it occurs (at least frequently). For example, imagine a simple three exon gene. Upon splicing exon 1 to exon 3, exon 2 would be excised as part of the intron lariat and subsequently degraded. So in order for exon 2 to be spliced to exon three you would need to either have splicing between exon ...


11

After performing a quick literature search, I am, as with GWW, unable to provide any literature against this occurring, although this paper by Black (2005) states that exons in multi-exon pre-mRNAs are always maintained in order. Black DL. 2005. A simple answer for a splicing conundrum. Proceedings of the National Academy of Sciences of the United States ...


7

If the anti-sense transcript is correctly annotated and in the databases (a very very big if), then it will have a different name. For example, the mouse Msx1 anti-sense transcript has the RefSeq accession NR_027920 while the sense transcript of the same gene is NM_010835. In general, each transcript that is transcribed from a given locus has a different ...


6

A good question (if a little mixed up on transcription vs. translation!) AUG is not always the start codon, but whatever the codon is it will always code for Methionine (or fMet, but still a variation on Met), even if the codon codes for a different amino acid otherwise. A separate transfer RNA (tRNAi, the initiator tRNA) is used for the arrangement of ...


5

Yes, you can find mutations in the genomic DNA which affect splice acceptor sites. Wikipedia lists the following outcome: Mutation of a splice site resulting in loss of function of that site. Results in exposure of a premature stop codon, loss of an exon, or inclusion of an intron. Mutation of a splice site reducing specificity. May result in variation in ...


5

If your goal is to avoid regulatory elements, I would not believe any prediction program, since new regulatory elements are found every day. The best way would be empirical and just clone and infect with a mutated 3'UTR and see if your gene's regulation is perturbed. You could at least swap the entire 3'UTR with that from another gene to see if there are ...


5

This study in E.coli is useful in setting out some additional issues at play here, in a system where post-translational modifications are basically a non-issue. The authors see clearly a correlation between mRNA and Protein levels (Fig. 3C) and theorize that differences translation rate and protein turnover are responsible for differences in protein level ...


4

I found the link to a commercial product by Evrogen here: http://www.evrogen.com/technologies/normalization.shtml They claim their method is compatible with nextgen sequencing platforms: cDNA normalization using duplex-specific nuclease (DSN) is a highly efficient approach that can be applied for normalization of full-length-enriched cDNA (Zhulidov ...


4

The only information you are missing is a way to identify the splice sites. There are many ways of doing what you need. The simplest, assuming you are sure of the origins of the mRNA, is to use a BLAST flavor, either plain BLASTn or, even better, BLAT, to compare your mRNA sequence to the genome of interest. BLAT really should be all you need if the mRNA ...


3

The protocol you are using will not only leave the sample with rRNA but also non coding RNA. Many RNA protocols will separate mRNA by affinity of a carrier to the polyA tail. This protocol references an older paper that estimates that only 5% of RNA is mRNA. I'd be surprised if this ratio changed by more than 2-3 fold in drosophila. I assume that %age ...


3

Shigeta's got a point: the ribosome is latched onto the mRNA so those two are intrinsically linked. You're really asking whether the ribosome comes off first or whether the tRNA does, but it's actually the new polypeptide, which makes sense: The stop codon is recognized by a protein, the polypeptide chain release factor (RF), which triggers the ...


3

First of all, it is the transcript and not the gene that starts with an AUG. Also, there are actually quite a few transcripts that start with different initiation codons, they are just the exceptions rather than the norm. As for the need, you can think of START and STOP codons as punctuation. The AUG is read like the first (capital) letter of a sentence ...


3

RNA Pol doesn't worry about stop codons. Transcription termination can occur through the formation of a hairpin in the new RNA sequence, or through the action of Rho proteins. A lot of the time prokaryotes have polycistronic mRNAs, that is, mRNAs with multiple protein coding regions. The stop codons are detected during translation, so you will often have ...


3

Point to know : aminoacyl-tRNA binds to mRNA its not just t-RNA.. So if there is no Amino-acid there is no aminoacyl-tRNA of that aminoacid.. so if there is no aminoacyl-tRNA, the anticodon of tRNA doesn't form a bond with mRNA, so the protein production halts (until the Amino acid produced). If the protein is not formed within a certain long time, the ...


2

There are references in the literature to the phenomenon of "exon scrambling" which seems to be what you are asking about, but the prevailing view is that the evidence for this process, which comes from comparing EST sequences with genome sequences, can be explained by cloning artefacts occurring during EST characterisation. Certainly I agree that there is ...


2

As far as I understand it (and I'll preface this by saying that initiation is not my strongest point), but prokaryotes utilize the beautiful AGGAGG Shine-Dalgarno sequence. Usually around 8bp upstream of the start codon, it is this sequence that the prokaryotic ribosome seeks out to initiate translation. It does this through a complementary region in the ...


2

You should ask this question over at biostars.org -- and could use a bit more clarity. Do you actually have RNA-seq data that you want to use to find the exon/splicing structure of an mRNA? First step would be to use a splicing-aware aligner. A few free ones are: STAR TopHat GSNAP Let us know why that wouldn't do what you want when you re-ask this ...


2

As others have stated, splicing does happen cotranscriptionally but you cannot be actively transcribing the RNA while it is being transcribed. However, it is generally accepted that a transcript can be polyadenylated while the RNA polymerase is still transcribing - this is called the torpedo model. The distinction is that the transcript has been cleaved ...


2

It's slightly more complicated than the comments above indicate. Glover-Cutter, K et al. (2007) RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes. Nature Structural & Molecular Biology 15: 71-78 The authors present evidence that RNA polII pauses 0.5 - 1.5 kb downstream of the poly(A) site where ...


2

It is not true that the anticodon of an uncharged tRNA can't bind to the mRNA. Bacteria have a mechanism called stringent response. This response is complicated, here is a shorter and simplified explanation. Further informations can be found at the wikipedia pages linked in the text. If during translation a certain amino acid is absent, an uncharged tRNA ...


2

According to the Gilson Pipettman User Guide you can autoclave the following parts: Tip ejector, Tip holder and the connecting nut (have a look into the PDF linked above if you are not sure about the parts). All other parts can not be autoclaved. Gilson says the following conditions can be used (page 17 in the user guide): Autoclave for 20 minutes at 121°C, ...


1

If your data came from a single lab and a standard protocol gcrma normalisation is a good method to use (and comes with the affy R package); if the arrays are of U133A or HGU133plus2 types frozen RMA may be a good method to use. Finally you can check for and control for batch effects using ComBat and use the corrected expression data.


1

This is not an easy task, as there are many factors which you have to take in account. First there are intra-essay differences as slightly different conditions for each set of probes for the repetitions. Then, there are inter-assay differences, as differences in the array slides and so on, this paper ("Analysis of microarray gene expression data") goes into ...


1

After a couple months I got this working. I ordered the psuedouridine and 5 methylcytidine from Trilink. I also switched from the mMessage mMachine kit the the Megascript kit. Megascript has all the nucleotides separated while mMessage mMachine has them pooled. You have to dissolve Trilink nucleotides so that the concentration is the same as the natural ...


1

just off the top of my head... since the ribosome is made of 2 large complexes which assemble and clamp onto the mRNA, I'd say it was the tRNA first, then the ribosome and mRNA would detach simultaneously.


1

I'd make a comment if I could, but I don't think mRNA ever enters the ER. When a ribosome is building a protein destined for export or for being inserted into the membrane, it travels to the ER membrane and binds to a protein in that membrane called Sec61, which guides the new peptide sequence through the membrane. However the portion of the ribosome that ...


1

The proteins mediating the transport of mRNA out of the nucleus (nucleoplasm/karyoplasm) are called "karyopherins" and belong to the nuclear pore complex family. Prior the creation of the final form of mRNA, various proteins are attached to the so called pre-mRNA and a mRNA-protein-complex is built (co-transcriptional). Some of these proteins carry factors ...


1

Helicos stopped shipping reagents around 2010-2011, as they couldn't compete with Illumina. For now it seems that reverse transcription -> Illumina-sequenced cDNA is so much cheaper than any other option, that's the method that is dominant. Oxford Nanopore has discussed direct RNA profiling, but unfortunately their first product still hasn't shipped (for ...



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