Sample collection for metranscriptomic sequencing

newBioinfo · 04-29-2013, 01:34 PM

Hi All,
I need to do metatranscriptomic sequencing from ice samples. I want to know how can samples be transported from the site to the lab for RNA isolation. The ice samples will be filtered on-site to remove unwanted elements, the filter will have all the microbes I need to study. In case of metagenomics the filter was preserved in ethanol and was transported to lab but I am wondering if I keep the filter in the ethanol all the RNA will be degraded.
Please can anyone suggest anything.

Thanks!!!

JackieBadger · 04-29-2013, 02:57 PM

You talk in the future then past tense...have you actually collected the samples?
If not, then do not preserve in ethanol. You need RNAlater. Although RNAlater is expensive, the recipe is not complicated and you can make your own for much cheaper.

newBioinfo · 04-29-2013, 03:21 PM

Quote:

Originally Posted by JackieBadger

You talk in the future then past tense...have you actually collected the samples?
If not, then do not preserve in ethanol. You need RNAlater. Although RNAlater is expensive, the recipe is not complicated and you can make your own for much cheaper.

Thanks JackieBadger,
I did some 16S rDNA sequencing from the samples collected from ice and they were preserved in ethanol. But, now I need to do metatranscriptomic for ice samples and I am not sure how to transport the filters with microbes from site to lab for RNA isolation.
The samples are not not collected yet for metatranscriptome.

Thanks!!!

JackieBadger · 04-29-2013, 04:41 PM

for transcriptome work you need to preserve as much RNA as possible...ethanol just won't do. Like I mentioned, you need to use RNAlater or make some of your own

newBioinfo · 04-30-2013, 09:25 AM

Quote:

Originally Posted by JackieBadger

for transcriptome work you need to preserve as much RNA as possible...ethanol just won't do. Like I mentioned, you need to use RNAlater or make some of your own

Thanks a lot!!!

Cofactor Genomics · 04-30-2013, 11:34 AM

Hello NewBioinfo,
I agree with JackieBadger on taking these steps to do all you can for the preservation of those RNA samples.

What becomes the real determinant is that assessment (QC) that you make of the RNA post isolation and prior to any library constructions (ie well before it gets anywhere close to a sequencer).

There are additional members on this board that have done thousands of RNA isolations and QC prior to sequencing. Our respective team has learned quite a bit over the past 5 years on what are the requirements for RNA to produce a successful sequencing product (RNA-seq is the number one project type initiated with our teams for 5 years in a row).
Below is a link to what our team is looking for concerning input quality/quantity to execute a successful project. I include it in hopes it in hopes it is useful:

http://www.cofactorgenomics.com/site...nce%20card.pdf

My best.

Jarret Glasscock
Cofactor Genomics
http://www.cofactorgenomics.com

Jarret Glasscock
Cofactor Genomics
http://www.cofactorgenomics.com

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04-29-2013, 01:34 PM	#1
newBioinfo Member Location: US Join Date: Mar 2012 Posts: 36	Sample collection for metranscriptomic sequencing Hi All, I need to do metatranscriptomic sequencing from ice samples. I want to know how can samples be transported from the site to the lab for RNA isolation. The ice samples will be filtered on-site to remove unwanted elements, the filter will have all the microbes I need to study. In case of metagenomics the filter was preserved in ethanol and was transported to lab but I am wondering if I keep the filter in the ethanol all the RNA will be degraded. Please can anyone suggest anything. Thanks!!!

04-29-2013, 02:57 PM	#2
JackieBadger Senior Member Location: Halifax, Nova Scotia Join Date: Mar 2009 Posts: 381	You talk in the future then past tense...have you actually collected the samples? If not, then do not preserve in ethanol. You need RNAlater. Although RNAlater is expensive, the recipe is not complicated and you can make your own for much cheaper.

04-29-2013, 04:41 PM	#4
JackieBadger Senior Member Location: Halifax, Nova Scotia Join Date: Mar 2009 Posts: 381	for transcriptome work you need to preserve as much RNA as possible...ethanol just won't do. Like I mentioned, you need to use RNAlater or make some of your own

04-30-2013, 11:34 AM	#6
Cofactor Genomics Registered Vendor Location: St. Louis Join Date: Jan 2010 Posts: 52	Hello NewBioinfo, I agree with JackieBadger on taking these steps to do all you can for the preservation of those RNA samples. What becomes the real determinant is that assessment (QC) that you make of the RNA post isolation and prior to any library constructions (ie well before it gets anywhere close to a sequencer). There are additional members on this board that have done thousands of RNA isolations and QC prior to sequencing. Our respective team has learned quite a bit over the past 5 years on what are the requirements for RNA to produce a successful sequencing product (RNA-seq is the number one project type initiated with our teams for 5 years in a row). Below is a link to what our team is looking for concerning input quality/quantity to execute a successful project. I include it in hopes it in hopes it is useful: http://www.cofactorgenomics.com/site...nce%20card.pdf My best. Jarret Glasscock Cofactor Genomics http://www.cofactorgenomics.com Jarret Glasscock Cofactor Genomics http://www.cofactorgenomics.com