[DIYbio] Isolation and sequencing of RNA

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?