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Old 04-04-2017, 11:10 AM   #1
renuka22
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Default Poly A and RNA seq

Hello,

I have some polysomal RNA samples, and I need to send them for polyA isolation and RNA seq (polyA selection). Does anyone know any good companies that can do polyA isolation and further sequencing? Only problem is I have only around 1.5 ug of polysomal rna remaining as I had sent few samples earlier as well but the company was not able to do polyA and the samples were wasted. Should this concentration be enough to get polyA?
Any suggestions/help will be really appreciated..

Thanks
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Old 04-12-2017, 11:35 AM   #2
pmiguel
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Quote:
Originally Posted by renuka22 View Post
Hello,

I have some polysomal RNA samples, and I need to send them for polyA isolation and RNA seq (polyA selection). Does anyone know any good companies that can do polyA isolation and further sequencing? Only problem is I have only around 1.5 ug of polysomal rna remaining as I had sent few samples earlier as well but the company was not able to do polyA and the samples were wasted. Should this concentration be enough to get polyA?
Any suggestions/help will be really appreciated..

Thanks
Of course 1.5 ug would be enough. But as discussed in the previous thread the amount of RNA in those samples could have been zero due to residual phenol in your samples confounding the nanodrop readings.

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Old 04-14-2017, 04:31 PM   #3
luc
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Perhaps a stupid question? The mRNA content in polysomal RNA samples should be very high. Why is a poly-A enrichment still needed?
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Old 04-17-2017, 06:21 AM   #4
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Perhaps a stupid question? The mRNA content in polysomal RNA samples should be very high. Why is a poly-A enrichment still needed?
I've never isolated polysomal RNA, so I don't know -- but from some details given on this site it looks like 3+ribosomes would be associated with each message. That is about 8000 bases of rRNA x 3 = 24,000 bases of ribosome per message. So it seems like the amount of ribosomal RNA would actually be quite high. >90% if we presume that this sites numbers would be representative of the average length of mammalian messages (2.2 thousand bases).

The OP didn't specify their methodology -- hopefully not sucrose density ultricentrifugation as that seems like it would need very large numbers of cells. In the video in the site I link to above, those look like 150mm petri dishes and the protocol specifies that they be grown almost to confluence. But since the OP was apparently isolating RNA from rat retinal ganglia I would expect yields to be very low unless they were pooling cells from dozens of animals.

Standard RNAseq methods would likely not suffice to generate usable libraries from the limited amount of RNA present in such a sample. So it is likely that some sort of low-input RNA method would be needed.

As long as researchers still blithely trust UV absorbance of a solution at 260nm to provide them with accurate quantitation of nucleic acids there is going to be time and money wasted attempting to sequence dilute solutions of phenol.

I personally find the situation particularly irksome because even a cursory glance at the UV spectrum -- and specifically at whether absorbance is higher at 270nm than at 260nm (the hallmark of phenol's spectrum near neutral pH) -- is enough to warn the researcher that UV spec isn't going to give them a usable answer as to the concentration of nucleic acids in solution.

If you teach molecular methods I beg you to hammer this point home to all of your students instead of the nearly useless 260/230 and 260/280 metrics.

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Old 04-17-2017, 01:13 PM   #5
renuka22
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Hello,

Perhaps people don't know much about polysomal RNA...

pmiguel - Yes you are right. Polysomal RNA is rich in rRNA. We have not used sucrose gradient due to yield issues and after doing polyA we hardly get anything. mRNA is just 1-3% of total RNA or here polysomal RNA. Thanks..
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Old 04-17-2017, 02:26 PM   #6
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Standard poly-A capture RNA-Seq protocols can go down to about 100 ng of Total RNA starting material these days. Kapa (I don't work for Kapa, but I do like their NGS kits) sells a mRNA HyperPrep kit that can supposedly go down to 50 ng of Total RNA. https://www.kapabiosystems.com/produ...yperprep-kits/

Any lower than that and I would recommend using something like the SMART-Seq v4 ultra low input RNA-Seq kit to capture poly-adenylated messages and generate cDNA and Nextera XT to create your sequencing library. Both of these kits are quite user friendly.
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Old 04-17-2017, 08:28 PM   #7
renuka22
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jteeee2 - Thanks for your suggestion..I have some samples for which the bioanalyzer is not able to detect any rna., might be there is no rna or very less. Please find attached the results from the company, for which the polyA library failed as they said the library amplification failed, and sequencer was not able to detect anything for sequencing. Is there a possibility that a more sensitive method will be able to detect the polyA mrna?
Attached Files
File Type: pdf QD157 Library issue report.pdf (581.5 KB, 24 views)

Last edited by renuka22; 04-17-2017 at 08:31 PM.
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Old 04-17-2017, 10:29 PM   #8
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Bioanalyzer traces indicate presence of RNA and perhaps using Pico Chip would have been given better profile. Most polyA capture kits assumes 3% polyA RNA in the sample and this is the minimum input for standard RNA-Seq library prep kits. It is possible that polyA RNA quantity in your samples were well below that threshold.

Best options would be the one suggested by jteeee2 or SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian if your species is mammalian and your experiment does not need actual polyA start sites.
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Old 04-18-2017, 12:10 AM   #9
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Default QuantSeq for low-input, p(A) specific gene expression analysis

Hello,
if only genes (and not transcripts) need to be identified, then the QuantSeq 3' mRNA-Seq kit provides a low-input (down to 0.1 ng total RNA input), highly poly(A) specific solution. The REV version also identifies poly(A) sites at the nucleotide level.
Highly validated (> 20 publications since the launch 2.5 years ago) and cost-effective (from USD 19.80 / lib prep), trial kits are available.
If you are interested, please contact us at info@lexogen.com. We also offer 3' mRNA-Seq DGE as a service.
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Old 04-18-2017, 06:17 AM   #10
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I agree with nucacidhunter about using the Pico RNA assay on the Bioanalyzer. It would be interesting to see what this material looks like on the more sensitive assay.

Has this material been column-cleaned/DNase treated? It looks a little odd, and there has been some conversation about phenol contamination.

If you want to get a real answer about whether there is RNA present or not, set up some qPCR experiments where you probe some transcripts that should be translated under your conditions (Actin, Gapdh, etc.) Use some Total RNA from the same tissue or cell line as a control. If you can't detect transcripts by qPCR, you are definitely not going to find them in your RNA-Seq experiment.
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Old 04-18-2017, 09:21 AM   #11
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jteeee2 - Yes I am planning to do qPCR on the samples to confirm. No, I have not done Dnase treatment, I did not want to go through extra treatments due to yield issues. Thanks for the suggestions. I will keep posted on the qPCR results..
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Old 04-18-2017, 11:44 AM   #12
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Quote:
Originally Posted by nucacidhunter View Post
Bioanalyzer traces indicate presence of RNA and perhaps using Pico Chip would have been given better profile. Most polyA capture kits assumes 3% polyA RNA in the sample and this is the minimum input for standard RNA-Seq library prep kits. It is possible that polyA RNA quantity in your samples were well below that threshold.
I don't think that was a bioanalyzer run on polyA+ RNA -- it was the total RNA.
The lack of any large or small rRNA subunit RNA peaks suggest either the stuff we are seeing there is not even RNA or something in the prep has interfered with the resolving that RNA.

At this point, either removing the phenol and checking concentrations on the preps again. Or, better, just firing up a fluorimeter with a high sensitivity RNA fluor to find out how much RNA, if any is present in the preps seems like the easiest course of action to me.

Unless you have some conversion metric between qPCR results and total RNA amounts, a positive qPCR basically tells you nothing since it is many, many orders of magnitude more sensitive than library construction/sequencing.

Bioanalyzer pico RNA chips are so finicky we basically gave up on using them. If the nano chip can't resolve this stuff, a pico chip will definitely not be able to.

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Last edited by pmiguel; 04-18-2017 at 11:54 AM.
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Old 04-18-2017, 08:38 PM   #13
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I will be doing high sensitivity assay on qubit. No idea if it will detect anything. But a company has agreed to do rna seq if the samples are >20pg. They mentioned they have a kit which can be sensitive for that less concentration. I am not sure though how the sequencing will look like with such a less concentration.
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Old 04-19-2017, 12:31 PM   #14
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The rule-of-thumb is 1 pg ~= 1 million RNAs of an average length of 2000 bases. So >20pg is a reasonable cut-off if you are looking to get ~10 million unique reads from a library and have a kit that is very efficient at converting RNA into amplicons.

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Old 05-12-2017, 09:19 AM   #15
renuka22
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Is it a good idea to pool 2 rat litters together to get more RNA and solve the yield issue? Will it affect the RNA sequencing data?
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Old 05-16-2017, 12:29 PM   #16
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Hi Renuka22,
What is your "yield issue"? Last I remember you were going to determine your actual yield using a fluorimeter. How much RNA does a rat yield you?

Since there are methods to generate libraries from single cells, you might want to use one of those and spare yourself all that dissection.

But if you do want to pool RNA from multiple litters, it will have pretty much the effects you might guess. You will likely get more RNA, but your sequencing results will probably have more noise in it. Whether that noise will overwhelm the signal of your experiment would depend on your experiment.

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