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8

A pre-tRNA is transcribed from tRNA genes in DNA by RNA polymerase III. Processing occurs in the nucleus, where a 5' sequence is cleaved by RNase P, the 3's CCA motif is added, and ~10% of the nucleotides are substituted. The tRNA are transported out via the pore complexes. Aminoacyl-tRNA synthetase enzymes attach amino acids in the cytoplasm in a 2-step ...


5

I think it is relatively unlikely that the RNA will degrade under this conditions. For the future, I would handle this differently: You can centrifuge the cells and snap freeze them in an appropriate buffer in liquid nitrogen and then store them at -80°C. I would not freeze the dry pellet, as these are often hard to re-dissolve after freezing. ...


4

As far as I know this has never been thoroughly analyzed for RNA, but there is an excellent paper on the precipitation of DNA and the usual conditions in BRL Focus by Zeugin (see below for the paper). Since DNA and RNA are pretty much the same (except for one OH-Group) and the conditions used for precipitation are also similar, I think we can use this for a ...


3

Yes, transcription of a polyA sequence will be unreliable: for a repetition of 15 times the same nucleotide, the rate of transcription errors will be huge. For example in E. coli, this paper http://www.ncbi.nlm.nih.gov/pmc/articles/PMC331007/ gives some order of magnitude of transcription errors caused by repeats of nucleotides. According to their data, for ...


3

I'm assuming that you have a solution of bacteria in your buffer directly after growing them for some time, and without performing a lysis step yet. The 2% SDS step looks like it is for lysis of your cells. In that case, centrifuging the bacteria and resuspending them in lysis buffer is a very common step in many protocols. Separating whole bacteria from ...


2

PBS is isotonic to most cells, so in this buffer your bacterial cells are intact. Centrifuging them to remove PBS won't harm them it is quite standard procedure. You have to be careful after adding 2% SDS because it will lyse your cells and then the RNA contained within them will become "free" and RNAs are fragile molecules.


2

Some general aspects about miRNA nomenclature in addition to canadianer's answer: mir refers to the pre-miRNA whereas miR refers to the mature form mir-x-y mir-x-z for example mir-1-1 and mir-1-2 refer to different precursors that give rise to same mature miRNA (i.e. miR-1). These different precursors can be regulated differently. The mature miRNA ...


2

The three RNA polymerases (RNAPs) are very similar to each other, yet not identical. As described in this article, there are subunits that are specific for each type of polymerases. In addition to providing unique substrates that polymerase-specific subunits bind to give each of the RNAPs their specific functionality, the two largest subunits also shape ...


1

Thermo Fisher has a protocol for the separation of host mammalian RNA from prokaryotic RNA, optimized for E. coli vs human, mouse or rat sequences. Capture oligonucleotides bind portions of the mammalian RNA and hybridize. These hybridized oligo/RNA are then removed from solution via "oligonucleotide-derivatized magnetic beads," after which the remaining RNA ...


1

let-7 is a miRNA family (let stands for lethal) and, in fact, was one of the first to be discovered (in Caenorhabditis elegans). let-7a is but one of many members of the family, with orthologs in many species. It seems rare in biology that established names are changed to match newer nomenclature, though I can't say for sure if that is why many (but not all) ...



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