Cloning a Bacterial mRNA

[Bluenoise]

Okay, lets say you isolated a bacterial mRNA through the binding of another RNA of much smaller size (say 70bp) how would you go about cloning and sequencing this bacterial mRNA you isolated?

 

Rules:

1) Bacterial RNA does not have a poly A tail.

2) You're completely clueless about the sequence of this mRNA you've isolated.

3) Your sample is contaminated by this small RNA by thousands if not millions of times the copy number of the RNA you're trying to isolate.

4) There are thousands of possible binding partners for this small RNA so you can't just search for the anti-sense.

 

So you basically have to selectively clone this mRNA and sequence it.

I've thought of a possible way to do it. But I want to hear someone else's suggestion first.

[CharonY]

Just off the top of my head:

depending on size differences between isolated mRNA and the small RNAs:

-denaturing PAGE and then reisolation of the right band

-chromatography (e.g. LC; nano-LC)

 

if sequence of small RNA known:

-make biotinylated oligos with the sequence of the small RNA, couple it to magnetic beads and fish the desired mRNA by magnetic separation

 

follow a cDNA normalization procedure (still needs screening, though)

 

That's basically what I can come up with in the spur of the moment. For better ideas I need a coffee, first.

 

(so what's your plan? )

[Bluenoise]

Actually I think we misunderstand each other. You're idea about coupling the small RNA to magnetic beads and fishing out the other one is what has already been done prior.

I'm taking about what's to be done after that.

 

IE. How would you go about making the cDNA from the mRNA you have captured. Even after eluting from the beads it will still be at a much lower level than the small RNA (some will fall off the beads), say a million times lower.

 

Anyways here's my idea. RT pcr using a random primer with a small over hang for say 5 cycles. Then add a primer complementary to this over hang and amplify for 20 cycles more. Run it on a gel excise a region say 200-300 bp larger than the small RNA (as the random primer products will have to be smaller than the template), reamplify it followed by TA cloning and sequencing. The reamplification is necessary because the smaller cDNA's created from the high copy number small RNA will amplify far better than the longer ones from the mRNA.

 

God I hope that makes sense. It's been a long week.

I wish prokaryotes had poly AAAA tails.

[foodchain]

Actually I think we misunderstand each other. You're idea about coupling the small RNA to magnetic beads and fishing out the other one is what has already been done prior.

I'm taking about what's to be done after that.

 

IE. How would you go about making the cDNA from the mRNA you have captured. Even after eluting from the beads it will still be at a much lower level than the small RNA (some will fall off the beads), say a million times lower.

 

Anyways here's my idea. RT pcr using a random primer with a small over hang for say 5 cycles. Then add a primer complementary to this over hang and amplify for 20 cycles more. Run it on a gel excise a region say 200-300 bp larger than the small RNA (as the random primer products will have to be smaller than the template), reamplify it followed by TA cloning and sequencing. The reamplification is necessary because the smaller cDNA's created from the high copy number small RNA will amplify far better than the longer ones from the mRNA.

 

God I hope that makes sense. It's been a long week.

I wish prokaryotes had poly AAAA tails.

 

 

Not to derail your thread, but do you study the mutation rates of such in variable environments by any chance? I would offer to help but my only advice that I can muster is it just sounds like a lot of hard work.

[Bluenoise]

I'm sorry but I'm unsure which mutation rates you're referring too. But no I do not study mutation rates.

[muchado]

Anyways here's my idea. RT pcr using a random primer with a small over hang for say 5 cycles. Then add a primer complementary to this over hang and amplify for 20 cycles more. Run it on a gel excise a region say 200-300 bp larger than the small RNA (as the random primer products will have to be smaller than the template), reamplify it followed by TA cloning and sequencing. The reamplification is necessary because the smaller cDNA's created from the high copy number small RNA will amplify far better than the longer ones from the mRNA.

 

God I hope that makes sense. It's been a long week.

I wish prokaryotes had poly AAAA tails.

 

 

I was going to suggest something along this line..random primer extension...its sounds like the most simple approach. There is a paper from David Bartel's lab in MIT ...check their protocols...here is the reference for the paper...Science vol 294 no. 5543..oct 26 2001 pg. 858-862. There lab web site once had this protocol for cloning miRNA..http://web.wi.mit.edu/bartel/pub/protocols/miRNAcloning.pdf might give you some idea.

 

Cheers!

[CharonY]

Ah, regarding the fishing idea, I omitted that I would have used biotinylated DNA, so that I can digest them away with DNAse.

I was very unclear and imprecise. Sorry about that (lack of coffee and such).

 

Your approach probably would als work, but wouldn't the small RNAs also be reamplified at a higher rate?

Personally I prefer to recover DNA of specific lengths with nano-LC then to reisolate them from a gel, but that is down to personal preference (and equipment).

[Bluenoise]

Well no because you have a size selection for the large RNA's.

 

Hmmm I guess I could use LC to size separate them, seems a little excessive though.

[CharonY]

Hmm i probably don't get it. Lemme see if I understood you:

First you add a random primer with a 5`tag for RT.

Result are random cDNA fragments from long and short RNAs with a tag.

Due to the vast amount of small RNAs probably the majority of the cDNA will have a small RNA origin, unless they are lacking the random primer binding site.

Then you add a primer complementary to the tag and make another one sided PCR.

Am I already missing something here? Because I do not see how the additional PCR step helps to select for the larger fragments. Or was your plan just to get enough cDNA and simply separate it on gel?

 

In that case I'd either use a polyA tailing reaction to the RNA to get polyA mRNA, or use a terminal to label the cDNA. That way you can use two primers to increase your cDNA yield and at the same time increase your chance to get your full mRNA sequence.

[Bluenoise]

After the first amplification you run the PCR products on a gel. You excise a region far larger than the small RNA. This way you've eliminated any PCR product that resulted from the small RNA. Next you amplify with the overhand to get enough insert to clone.

[CharonY]

D'oh, I see now. One small problem is possibly that even at a higher band a low amount of small cDNA might be co-isolated (agarose gel are not that stringent in seperation). That kind of contamination usually doesn't hurt for direct cloning, though they might get co-amplified in PCR.

But that protocol is short enough to be worth a try nonetheless

[Bluenoise]

I know that has me worried a little bit, DNA runs in a Gaussian distribution on gels unfortunatley. But hopefully the effect isn't that large.

Yeah seems like a nice easy protocol I can get an undergrad to do in a day .