Isolation and sequencing of RNA

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Luke

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Mar 19, 2015, 11:29:09 PM3/19/15
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Hi Guys

 

First of all sorry for my bad English. I should do a final dissertation in my school. (Fortunately not in EnglishJ) My goal is to isolate and sequencing an RNA-Strand of a cell. I have development a little plan, but I don`t know if he works and would be happy about some assessments.

The plan:

Step 1: I would isolate the RNA with a RNA Isolation Kit. Which one, I don`t know yet, probably the cheapest one J Suggestions?

Step 2:

I would change this RNA-Fragments to cDNA. (With reverse Transcriptase)

 Step 3:

To pick up only one RNA-Fragment, I would use Agarose gel. So I let all these DNA-Fragments walk through the gel and so they separate and I can pick up one. (The concentration of Agarose must be very high, so also the RNA-Fragments with the difference of only one nucleotide would separate.  I don`t know if this works and would like other suggestions…

Step 4:

I would copy the DNA with PCR

Step 4: Sequencing

I had the idea to do it like in this video:

http://www.gizmag.com/ion-proton-sequencer-decodes-dna/21092/

So if I had a big concentration of the same cDNA I could notice the difference between the PH with a “normal” (not very exactly) PH-Meter. One of the things that could be a problem is to separate the cDNA with the free nucleotides after every Step. I thought on a filter or with blotting. (But is this possible in a school laboratory?)

At the school it has a PCR-Machine, a Machine for the Gel electrophorese and a centrifuge.

 

So if you could say if it`s possible and can give some advice, I would be very thankful.

 

Kind regards

Luke

 

The calculation, if a normal PH-Meter could notice the PH-Difference:

H3O+ Concentration at PH 7: 10^-7 mol/l

H3O+ Concentration at PH 6,9: 1,26 * 10^-7 (6,9 =-log(c(H3O+)))

Difference of the H30+ Concentration:

1,26*10^-7-10^-7=2.6*10^-8 mol/l

Absolute amount:

2,6*10^-8 mol *6*10^23 = 1.56 * 10^16

The Thermocycler copy the cDNA exponentially:

2^x=1,55*10^16,  x = 54

So if you let the Thermocycler copy the DNA 54 times and the PH-Meter can notice the difference of 0.1 PH, it works?

Luke

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Mar 22, 2015, 2:47:51 PM3/22/15
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Any help???

Nathan McCorkle

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Mar 22, 2015, 10:41:27 PM3/22/15
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On Thu, Mar 19, 2015 at 1:51 PM, Luke <lukas.lu...@gmail.com> wrote:

> So if you let the Thermocycler copy the DNA 54 times and the PH-Meter can
> notice the difference of 0.1 PH, it works?

Some things that immediately come to mind relate to the ionic strength
of the solution. Also, you'd need to measure the pH before adding free
nucleotides, as these will contribute as acids I think. Measuring
low-concentration DNA solution, then adding master mix and amplifying,
then purifying seems like opportunity for lots of transfer loss to
affect the results. Not to mention how much the ion-selective
electrodes (ISE) tend to drift... you'd also need a way to ensure the
ISE didn't cross-contaminate your samples unless you weren't
particularly interested in using them.

I wonder how you would do with a visual pH indicator, but at that
point you're just trading the use of something like 260nm light for
something higher in the visible (and it's less direct indication).

In theory it sounds like a reasonable idea to try out... but I would
recommend doing a good prior-art literature search as it does seem
like something someone could have tried before.

Should you try this, let us know the results of course!!!

Jeswin

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Mar 23, 2015, 12:39:08 PM3/23/15
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From what I understand from your question, you would like to test the principle behind the Life Technologies Ion Proton Next-gen-sequencing. May I ask why you want to perform this test on RNA? One thing I notice is that you would need a high concentration of sample DNA (of a specific size) in order for your pH meter to detect. Therefore, wouldn't working with plasmid DNA be a better choice? It's easier to work with.

Secondly, it might be difficult unless you get a pH meter that can get results from low volumes and has high precision. Or, you may have to devise an indirect way to measure this.

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Luke

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Apr 3, 2015, 1:46:22 PM4/3/15
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Thanks for the help. I will do it and of course let you know the results. I will thinking about the problem with the ISE and hopefully find a solution. And yes DNA Plasmid could be a better choice, I will discuss it with the others of my group.

Luke


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