16S rRNA Extraction

[Makky]

Hi all

In fact I need a help. I need a good method and speed one to extract 16S rRNA from bacteria.

 

Thanks,:eek:

[ecoli]

Do you need to keep it as RNA?

 

If not, I recomend doing an RNA isolation, and reverse transcription to turn it into cDNA. That way you can use 16s RNA primers (I have the sequence somewhere, if you need it) to isolate just the expression of 16s.

[CharonY]

also the question is whether you really need purified 16srRNA. Most applications just use total RNA (a separation of mRNA and rRNA is possible, but tricky and there will be losses).

Generally the fastest way is one of the gazillion total RNA kits (e.g. from Qiagen or Ambion).

Depends on the budget, though.

[jerrywickey]

Just a dumb question.

 

Is the useful side of cDNA reversed 5' to 3'?

 

Is ribosomal RNA transcribed from the same side of DNA as mRNA? I think it would have to be.

 

If so, how would a cDNA transcription of 16s rRNA be different from the DNA template from which the rRNA was originally transcribed? Or is sequencing the 16s rRNA needed in order to find the DNA template among possible splices? Because the cDNA transcription of the rRNA would not have the introns?

 

Sorry for the dumb question. Its just not sitting right in my head for some reason.

 

Jerry

[ecoli]

during transcription, the coding strand is read 3'-5' and RNA is made 5'-3'. I think during RT, the cDNA is copied from 5'-3'... not sure about that, though. I'll check my biochem textbook tomorrow if CharonY hasn't already given you the right answer by then.

[jerrywickey]

Thanks I will look for other answers too.

 

Jerry

[CharonY]

Ah, it is quite common that one gets confused about the directions of the DNA coding strand. However, a simple way to memorize it is that the polymerases move from 5'->3'. The reverse transcription is no exception, it is simply a RNA dependent DNA polymerase.

Transcription of rRNA genes works precisely the same way as for mRNa, only because it is a structural RNA does not change the transcription method.

Regarding the sequencing reaction:

first note that due to technical reasons you usually do not sequence a single strand. Normally after the RT you amplify the ssDNA by PCR and thus yielding dsDNA again. However, the direction of sequencing (whether you are on the codogenic strand, or not) is dependent on the primer that you use.

 

So if you omit a PCR step and directly sequence the single strands directly from the RT, you'd get the codogenic strand.

 

f so, how would a cDNA transcription of 16s rRNA be different from the DNA template from which the rRNA was originally transcribed? Or is sequencing the 16s rRNA needed in order to find the DNA template among possible splices? Because the cDNA transcription of the rRNA would not have the introns?

 

I gather from those questions that you ask why people actually bother to sequence the rRNA as opposed just to isolate DNA?

Let's start with the latter questions. Bacteria do not splice and do not have introns. So this is obviously out of the way. Now there are different reasons why you would be more interested in DNA instead of RNA. The most obvious one is that ribosomal DNA is more stable than the rRNA counterpart. As such by DNA profiling you will also find RNA genes of inactive organisms. Moreover, you can tag and separate rRNA of metabolically active organisms (e.g. via stable isotope probing), which you cannot with DNA.

[vbreemars]

First let me introduce my self.

 

My name is vbreemars and I from Indonesia. Nowadays I work as research assistant at biotechnology Lab. Bogor Agricultural University.

 

In my lab recently, I am doing research by 16s rRNA-based method to identify the species of our microbe isolate. But I do it by extracting the DNA genome and PCR it by specific primer (27F-1492R), is it okay?

 

I ask it because seemly you all do it by RNA extraction and doing RT-PCR on it.

 

Sorry if my grammar not good and if the question not proper?

[jerrywickey]

Cheron,

 

very helpful thank you

 

JErry

[CharonY]

To vbreemars,

 

just to clarify: 27F and 1492R are universal primers, these are conserved regions of the 16s rRNA gene. If they were specific primers you would only get amplificates from a single genus (or even species).

Anyway, as I mentioned above, both approaches are viable. RNA has a faster turnover, though, so microbes detected that way are more likely to be still active. What some do is to do both, used DNA and cDNA to analyse the differences between those two pools.

[vbreemars]

I'm sorry, I'm not get the point you try telling me.

 

We have several untitled culture of microbe at my lab. I want to know the genus or the species of the microbe. I doing it by extracting the DNA genome and amplify the specific region of 16s rRNA. Than I'll Blast the sequence to get the distance tree result. the purposes of my work is to identify the genus and species of our microorganism.

 

So what is the differences sequence data that gained from RNA and DNA. the sequence will give same result in blast and distance tree analysis, am i right??

 

regards.

[CharonY]

As I have mentioned above, rRNA is often isolated by microbiologists interested in the active microorganisms in the environment. RNA degrades much faster than DNA, so if you only go for the DNA chances are that you will also have many sequences of bacteria that are long dead. Everyone amplifying rRNA sequences will at some points notice that: if you do not work in a clean area you might get amplifications using universal rRNA gene primers even if no template is added (one of the reasons why these bothersome PCR cabinets are used).

If you isolate RNA on the other hand, chances are higher that you will get sill living organisms. In other words, you will get somewhat different results if you amplify directly from DNA as opposed to RT-rRNA. This is of course only the case in complex communities. If you got some highly enriched samples or even pure cultures it does not make sense to go for the RNA.

[vbreemars]

thanks very much i got the point

[mustkara]

Not sure the textbook will help but I used the textbook

 

RNA Methodologies: A Laboratory Guide for Isolation and Characterization, 3rd Edition

 

to study RNA and RNA isolation. Wish some help.