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Bacteria Lysis Reagent For RNA extraction


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12 replies to this topic

#1 Adrian K

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Posted 01 December 2009 - 12:27 AM

Hi all, I'm new in RNA extraction.

I am trying to extract bacteria RNA. However, protocols I had read so far requires TE buffer and lysozyme.
I was wondering if there is any other recipe for Bacteria lysis prior to RNA extraction (without involve lysozyme).

Also, alkaline lysis (NaOH +SDS) was used in plasmid extraction but I have yet to see it was being use in RNA extraction...is there any explanation for this? Thank you.

Awaits your suggestion.

Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

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"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

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#2 gogreen

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Posted 01 December 2009 - 12:33 AM

Hi Adrian

Alternatively you can try bead beating to break the cells and proceed the extraction..But in my experience, lysozyme is the better way to do it. What bacteria do u work on?

#3 Adrian K

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Posted 01 December 2009 - 12:39 AM

Hi gogreen,
I'm dealing with B.pseudomallei. Besides Beads beating, is there any chemicals which I can try on?

There is DNA lysis buffer recipe contain: "Triton-X 100, EDTA, Tris-cl"... will it work for RNA isolation?

Source: http://www.cbs.umn.e...ls/bactPCR.html

Edited by adrian kohsf, 01 December 2009 - 12:48 AM.

Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#4 gogreen

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Posted 01 December 2009 - 01:03 AM

Hi Adrian, Just googled to find that its a gram negative bacteria...so it might be easier to break the cells, you can give a try with the lysis buffer, try to do that at 65 degrees for 10 minutes with constant vortexing..My experience with bacterial RNA isolation methods is that most of them work, but the extent to which it works differs..The key is to lyse most of the cells in the suspension to get maximum yield. The lysis might not be very efficient in first place, you might have to do a lot a trials. Can I ask why you dont choose lysozyme or beads which are tried and tested methods and why u wanna try a new protocol?

Edited by gogreen, 01 December 2009 - 01:11 AM.


#5 molgen

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Posted 01 December 2009 - 01:59 AM

Adrian,
Have you tried any commercially available kit and fond it didn't work?

#6 Adrian K

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Posted 01 December 2009 - 08:46 PM

Hi gogreen & molgen,

The reason I try not to use lysozyme is because I have to prepare it fresh. I try to avoid the beads vortexing to avoid generate aerosol as my bacteria is quite dangerous and pathogenic. After the cell lyses I try to use Trizol method with column purification. I afraid that certain chemicals will degrade RNA and affect downstream process such as Real-time PCR.

My experience with Promega SV Total RNA Isolation System and GeneMATRIX Universal RNA purification Kit (Eurx) gives genomic contamination. Maybe I had load too much of cells in it, even after DNAse treatment (Promega) I still can't get rid of it. This is the reason I was try to use a trizol + column method as was suggested in the forum: http://www.protocol-...e...c=8032&st=0

Any comments or suggestions?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#7 gogreen

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Posted 01 December 2009 - 10:50 PM

hi Adrian,

I used to store Lysozyme for over a month after dissolving it in Tris...But you can also buy lysozyme in solution which can be stored quite easily. I would choose lysozyme over anything else for the ease of use and it works for any bacteria..
for the gDNA carryover, I think if you take only an aliquot of the RNA after quantification, say 5 ug for the DNase treatment, you can get much cleaner RNA than taking the whole thing (I was assuming that you process the whole thing)

#8 Adrian K

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Posted 02 December 2009 - 06:28 AM

hi Adrian,

I used to store Lysozyme for over a month after dissolving it in Tris...But you can also buy lysozyme in solution which can be stored quite easily. I would choose lysozyme over anything else for the ease of use and it works for any bacteria..
for the gDNA carryover, I think if you take only an aliquot of the RNA after quantification, say 5 ug for the DNase treatment, you can get much cleaner RNA than taking the whole thing (I was assuming that you process the whole thing)


Hi gogreen,
:) :) ;) :P :) :D Thanks for your idea of using only aliquot for DNase treatment...you are right, I process the whole thing...

You mention the lysozyme in solution which can be purchase and store. can you share with me the brand or product?

Thanks again.

Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#9 gogreen

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Posted 02 December 2009 - 06:35 AM

hi Adrian


Good to hear it was of some help! ;)
I remember seeing a lysozyme solution from sigma...but could not track that in their websit now...But here is one from Epicentre .. http://www.epibio.com/item.asp?ID=338

Edited by gogreen, 02 December 2009 - 06:35 AM.


#10 Adrian K

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Posted 02 December 2009 - 07:09 AM

Hi gogreen,

Thanks for your help. I glad to know a RNA expert like you.

Just one more thing...how you make your lysozyme buffer? got reference or protocol to share?

Thank you.

Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#11 gogreen

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Posted 02 December 2009 - 07:56 AM

Hi Adrian

I was using sigma lysozyme powder. I used to dissolve it in water...I remember one protocol recommending to dissolve it in 10/100mM Tris (I dont remember 10 or 100!..sorry) Make 10mg/ml solution, They say the 10mg/ml can be stored at 2-8deg for a month...I've stored even more without any loss in quality ...For most of the gram negative bacteria, a 10-15 min incubation at a final concentration of 1mg/ml lysozyme should give sufficient lysis

I got the sigma Lysozyme solution...the product catalog number is L3790 ...
All these stuff are given in the product details in the sigma website, just have a look there for more details :rolleyes:

Edited by gogreen, 02 December 2009 - 07:58 AM.


#12 Adrian K

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Posted 02 December 2009 - 02:32 PM

Hi Adrian

I was using sigma lysozyme powder. I used to dissolve it in water...I remember one protocol recommending to dissolve it in 10/100mM Tris (I dont remember 10 or 100!..sorry) Make 10mg/ml solution, They say the 10mg/ml can be stored at 2-8deg for a month...I've stored even more without any loss in quality ...For most of the gram negative bacteria, a 10-15 min incubation at a final concentration of 1mg/ml lysozyme should give sufficient lysis

I got the sigma Lysozyme solution...the product catalog number is L3790 ...
All these stuff are given in the product details in the sigma website, just have a look there for more details :)



Thanks GoGreen!!!!
You safe my life!!!!

Thanks and I'll let you know any out come as soon as my kits were available.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#13 gogreen

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Posted 04 December 2009 - 07:40 AM

Hey Adrian, its my pleasure...all the best...do update about the results :D




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