Reverse transcription of poly-A tailed miRNAs using anchored Oligo dT primers?&# - (Jan/25/2006 )

Hi all,

I trying to clone miRNAs using the following protocol:

1. miRNAs are isolated from tissue.
2. miRNAs are poly A tailed using poly-A-polymerase.
3. poly A tailed RNAs are reverse transcribed using an anchored oligo dT primer.
4. amplified by PCR
5. cloned into a plasmid
6. sequenced


step 1 and 2 work nicely. I can isolate my miRNAs and attach a poly A tail to them using poly A polymerase, however I don't get any appropriate cDNA via RT. My RT primer is the following:

5'-ATT GAT GGT GCC TAC AGT TTT TTT TTT TTT TTT TTV N-3'

The (T)18 part is supposed to anneal to the poly A tail just at the and of the miRNA via the anchor 5'-VN-3'. When I check my cDNA after RT, my products are far bigger than expected. The expected size would be something around 80nt, however my RT-products are around 500-1000 nt.
I know that the poly A tailed miRNAs are far bigger than 20nt plus a small poly A tail, yet the anchored RT-primer should still anneal right at the end of the miRNA.

Any ideas what's going wrong?
Would you suggest a primer that has a bigger anchor in the desired transcript? e.g.: --TTT VNN NNN -3'???

Your help would be appreciated. This RT is driving me crazy.

Thanks for your help.

tevi

Hi, here some refer for you:
For RT-PCR
Biotechniques. 2005 Oct;39(4):519-25.
For cloning:
Nucleic Acids Res. 2004 Mar 12;32(5):1688-95. Print 2004.
FEBS Lett. 2005 Jul 4;579(17):3849-54.
hope these help you
Rui

Based on your expected amplicon size, I'd feel that you might working on cloning of miRNA using method describe by the papers I mentioned,
Here I wonder your method for miRNA isolation, it is recommended using small size RNA (around 20nt) which could be separated by PAGE and recovered by elution…
Your RT-primer is OK although but I am not sure your forward primer sequence.
By the way, I’d prefer hot start Taq, and using PCR program similar for real time PCR, for instance: 60C for annealing (not 72C) for 1 min which is good for sized amplicons...

You can select for short products in the PCR stage by shortening the extension time. Probably you would need to use a two-step denature and combined anneal/extend reaction, and the rate of heating and cooling would be critical in selecting for very short fragments.

Base on your information, the expected miRNAs’ cDNA should around 40nt (24nt provided by RT-primer, and ~21 nt come from miRNA sequence), why you’ve stated 80nt as expected size for miRNAs’ cDNA?
Have you add 5’ adaptor before RT, or added SMART adaptor in RT reaction?
It seems that you have not perform PCR after RT reaction…

Dear rshi,

yes, I have a smart switch oligo that is supposed to elongate the transcript during the RT.
5'-ATC GTA GGC ACC TGA AAG GG-3'.
Subsequently the cDNA is supposed to be amplificated using the following primers:

Left:
5'-ATC GTA GGC ACC TGA AA-3'

Rigth:
5'-ATT GAT GGT GCC TAC AG-3'

Maybe there is something wrong about the smart switch oligo. The approach is basically adapted from the following protocol:

http://banjo.dartmouth.edu/lab/MicroRNAs/A...oRNAcloning.htm

Any ideas?

Thanks for your help.

Tevi

I am not sure whether the excess smart oligos can work on reverse transcript products of another smart oligos, for here the incubate time should be too much for small size transcript…

By the way, some RNAs resistant to PAP and end with GGG.. might function as smart oligo, but such products might not make you in great trouble at PCR step.

My suggestion: using your RT products for PCR directly, while under PCR condition for small size fragment (60C for extension not 72C 1 min), then check amplicon size first on agrose gel or PAGE. If necessary, you might cut amplicon band around desired size and re-amplify for following cloning step.

Or you might think about other strategy, such as add poly C to reverse transcript instead of smart oligo, however, it still suffer the same disadvantage of smart oligo strategy: precise 5' terminus of miRNA cannot be determined with certainty although most miRNA do not end with G at 5'.

Or You might think about the adaptor based cloning…